Glucagon analogs exhibiting gip receptor activity

ABSTRACT

Provided herein are glucagon analogs which exhibit potent activity at the GIP receptor, and, as such are contemplated for use in treating diabetes and obesity. In exemplary embodiments, the glucagon analog of the present disclosures exhibit an EC50 at the GIP receptor which is within the nanomolar or picomolar range.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/662,868, filed Jun. 21, 2012, and U.S. Provisional PatentApplication No. 61/716,878, filed Oct. 22, 2012; the contents of eachapplication are incorporated by reference in their entirety into thepresent application.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 427 kilobytes ACII (Text) file named“225596_SeqListing.txt,” created on Jun. 11, 2013.

BACKGROUND

According to the most recent data from the National Diabetes Fact Sheetof the American Diabetes Association, 23.6 million children and adultsin the United States are afflicted with diabetes. Each year, 1.6 millionnew cases of diabetes are diagnosed in people aged 20 years or older.According to a study recently published in the Journal of the AmericanMedical Association, over two-thirds of adults in the United States areeither overweight or obese (Flegal et al., JAMA 303 (3): 235-241 (2010))and over one third of this population is obese.

The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucosedependent insulinotropic peptide (GIP), are naturally-occurring peptidehormones. Both GLP-1 and GIP stimulate insulin synthesis and secretionin a glucose-dependent manner and do not produce hypoglycemia (see,e.g., Nauck et al., J. Clin. Endocrinol. Metab. 76:912-917 (1993) andIrwin et al., Regul. Pept. 153:70-76 (2009)).

GLP-1 has been shown to be effective as adjunctive therapy for diabetesand is associated with weight loss. What remains unclear aboutGIP-targeted therapy, however, is whether successful treatment ofdiabetes and obesity will be achieved through antagonizing the effectsof this hormone (e.g., via GIP receptor antagonism) or through mimickingor enhancing the effects of GIP.

SUMMARY

Provided herein are peptides that are GIP agonist peptides contemplatedfor use in treating subjects in need thereof, e.g. with diabetes andobesity.

Native glucagon does not activate the GIP receptor, and normally hasabout 1% of the activity of native GLP-1 at the GLP-1 receptor. In someembodiments, the peptides are glucagon analogs comprising a structurebased on the amino acid sequence of native human glucagon (SEQ ID NO: 1)but differing at one or more positions as compared to SEQ ID NO: 1,wherein the differences, or modifications, enhance the agonist activityof the analog at the GIP receptor. Such glucagon analogs will haveagonist activity at the GIP receptor greater than that of nativeglucagon and, in some aspects, greater than that of native GIP. In someor any embodiments, the GIP agonist has a GIP percentage potency of atleast 0.1%. In some or any aspects of the present disclosures, theglucagon analog additionally exhibits agonist activity at one or both ofthe glucagon receptor and the GLP-1 receptor. Accordingly, GIP agonists,GIP-GLP-1 co-agonists, GIP-glucagon co-agonists, and GIP-GLP-1-glucagontriagonists are provided herein.

In exemplary embodiments, the selectivity of the GIP agonist peptide ofthe present disclosures for the human GLP-1 receptor versus the GIPreceptor is less than 100-fold. In some or any embodiments, the GIPagonist peptide has GIP percentage potency within 20-fold or 10-folddifferent (higher or lower) of the glucagon percentage potency and/orGLP-1 percentage potency.

In exemplary embodiments, the glucagon analog comprising (i) an aminoacid comprising an imidazole side chain at position 1 (e.g., His), (ii)a DPP-IV protective amino acid at position 2, optionally,aminoisobutyric acid, (iii) an amino acid comprising a non-native acylor alkyl group, optionally at any of positions 9, 10, 12, 16, 20, or37-43, optionally wherein the non-native acyl or alkyl group is linkedto such amino acid via a spacer; (iv) an alpha, alpha disubstitutedamino acid or an amino acid comprising an imidazole side chain atposition 20, and (v) up to ten (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8 or 9)additional amino acid modifications relative to SEQ ID NO: 1.

In exemplary embodiments, the glucagon analog comprising (i) an aminoacid selected from the group consisting of: Ala, Leu, Ile, Val,norvaline, homoserine, Met, and methionine sulfone at position 3 of theanalog; (iv) an acylated or alkylated amino acid at any of positions 9,10, 12, 16, 20, or 37-43, optionally wherein the non-native acyl oralkyl group is linked to such amino acid via a spacer; (iii) an alphahelix stability amino acid at one or more of positions 16-21, and (v) upto ten (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, or 9) additional amino acidmodifications relative to SEQ ID NO: 1. In exemplary embodiments, whenthe glucagon analog lacks a hydrophilic moiety, the glucagon analogexhibits a GIP percentage potency of at least 0.1% (e.g., at least 1%,at least 10%, at least 20%). In exemplary embodiments, the glucagonanalog has less than 100-fold (e.g., less than 50-fold, less than25-fold, less than 10-fold) selectivity for the human GLP-1 receptorversus the GIP receptor. In exemplary embodiments, the glucagon analogexhibits an EC50 at the GLP-1 receptor which is within 100-fold (e.g.,within 50-fold, within 25-fold, within 10-fold) of its EC50 at the GIPreceptor.

Throughout the application, all references to a particular amino acidposition by number (e.g., position 28) refer to the amino acid at thatposition in native glucagon (SEQ ID NO: 1) or the corresponding aminoacid position in any analog thereof. For example, a reference herein to“position 28” would mean the corresponding position 27 for a glucagonanalog in which the first amino acid of SEQ ID NO: 1 has been deleted.Similarly, a reference herein to “position 28” would mean thecorresponding position 29 for a glucagon analog in which one amino acidhas been added before the N-terminus of SEQ ID NO: 1.

The invention also provides a peptide comprising

-   -   (a) the sequence of:

(SEQ ID NO: 66)HX₂X₃GTFTSDX₁₀SKYLX₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁FVQWLX₂₇X₂₈X₂₉X₃₀PSSGX₃₅PPPSwherein:

-   -   X₂ is AIB;    -   X₃ is Gln or an amino acid comprising a side chain structure of        Structure I, II, or III, as described herein;    -   X₁₀ is Tyr or an amino acid covalently attached to a C12 to C18        acyl or alkyl group;    -   X₁₅ is an acidic amino acid, optionally, Glu or Asp;    -   X₁₆ is any amino acid, optionally, any amino acid other than        Gly, Pro, and Ser; e.g., Glu, Ala, alpha, alpha-disubstituted        amino acid (e.g., AIB), His, Lys,    -   X₁₇ is Arg, His, Gln;    -   X₁₈ is Arg or Ala;    -   X₁₉ is Ala or alpha, alpha disubstituted amino acid;    -   X₂₀ is a selected from group consisting of: alpha,        alpha-disubstituted amino acid (e.g., AIB) or Gln or His, Lys,        or Ala;    -   X₂₁ is an acidic amino acid, optionally, Asp or Glu;    -   X₂₇ is Leu, Ala, Nle, or Met;    -   X₂₈ is Ala or an acidic amino acid (optionally, Asp or Glu);    -   X₂₉ is aliphatic, e.g., Ala or Gly or AIB or Val;    -   X₃₀ is small aliphatic amino acid, e.g., Ala or Gly    -   X₃₅ is Ala or a basic amino acid (optionally, Arg or Lys);

wherein, when X₂₈ is an acidic amino acid, X₃₅ is a basic amino acid;

optionally, wherein, when X₂₈ is Asp, then X₂₉ is not Gly

wherein, when X₁₀ is Tyr, the peptide comprises at position 40 an aminoacid covalently attached to a C12 to C18 acyl or alkyl group, and,wherein, optionally, the peptide comprises Gly at position 41,

wherein the C-terminal amino acid of the peptide is amidated; or

-   -   (b) the sequence SEQ ID NO: 66 with up to 3 (e.g., 1, 2, or 3)        amino acid modifications relative to SEQ ID NO: 66, wherein the        peptide exhibits agonist activity at the human GIP receptor and,        the human GLP-1 receptor and/or the human glucagon receptor.

The present disclosures also provides a peptide comprising

-   -   (a) the sequence of

(SEQ ID NO: 67)X₁X₂X₃GTFTSDX₁₀SKYLX₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁FVQWLX₂₇X₂₈X₂₉X₃₀PSSGX₃₅PPPSwherein:

-   -   X₁ is an amino acid comprising a side chain imidazole,        optionally, a His or His derivative;    -   X₂ is a DPP-IV protective amino acid, optionally, an alpha,        alpha-disubstituted amino acid (e.g., AIB);    -   X₃ is selected from the group consisting of Glu, Ala, Leu, Ile,        Nle, Val, NorVal, homoserine, Met, methionine sulfoxide,        methionine sulfone, acetyl-Orn, acetyl-diaminobutanoic acid,        acetyl-Lys;    -   X₁₀ is an amino acid covalently attached to a C12 to C18 acyl or        alkyl group;    -   X₁₅ is an acidic amino acid, optionally, Glu or Asp;    -   X₁₆ is any amino acid, optionally, any amino acid other than        Gly, Pro, and Ser; (e.g., Glu, Ala, alpha, alpha-disubstituted        amino acid (e.g., AIB), His, Lys)    -   X₁₇ is Arg, His, Gln;    -   X₁₈ is Arg or Ala;    -   X₁₉ is Ala or alpha, alpha disubstituted amino acid;    -   X₂₀ is a selected from group consisting of: alpha,        alpha-disubstituted amino acid (e.g., AIB) or Gln or His, Lys,        or Ala;    -   X₂₁ is an acidic amino acid, optionally, Asp or Glu;    -   X₂₇ is Leu, Ala, Nle, or Met;    -   X₂₈ is Ala or an acidic amino acid (optionally, Asp or Glu);    -   X₂₉ is aliphatic, e.g., Ala or Gly or AIB or Val;    -   X₃₀ is small aliphatic amino acid, e.g., Ala or Gly    -   X₃₅ is Ala or a basic amino acid (optionally, Arg or Lys);        wherein, when X₂₈ is an acidic amino acid, X₃₅ is a basic amino        acid;        optionally, wherein, when X₂₈ is Asp, then X₂₉ is not Gly        wherein, when X₁₀ is Tyr, the peptide comprises at position 40        an amino acid covalently attached to a C12 to C18 acyl or alkyl        group, and, wherein, optionally, the peptide comprises Gly at        position 41,        wherein the C-terminal amino acid of the peptide is amidated; or    -   (b) the sequence SEQ ID NO: 67 with up to 3 amino acid        modifications relative to SEQ ID NO: 67, wherein the peptide        exhibits agonist activity at the human GIP receptor and, the        human GLP-1 receptor and/or the human glucagon receptor.

In exemplary aspects of the peptides comprising the sequence of SEQ IDNO: 66 or SEQ ID NO: 67 with up to 3 (e.g., 1, 2, or 3) amino acidmodifications, X₂₉ is not Gly when X₂₈ is Asp. In exemplary aspects ofthe peptides comprising the sequence SEQ ID NO: 66 with up to 3 (e.g.,1, 2, or 3) amino acids, relative to SEQ ID NO: 66, (i) X₁ is an aminoacid comprising a side chain imidazole, (ii) X₂ is a DPP-IV protectiveamino acid, optionally, an alpha, alpha-disubstituted amino acid, (iii)the C-terminal amino acid of the peptide is amidated, and (iv) thepeptide comprises at position 40 an amino acid covalently attached to aC12 to C18 acyl or alkyl group, when X₁₀ is Tyr, and optionally, thepeptide comprises Gly at position 41.

In exemplary embodiments, the GIP agonist peptides comprise an aminoacid sequence of any of SEQ ID NOs: 19, 21-26, 28, 287, 289-294, 296,520, 522-527, and 529, or any of SEQ ID NOs: 30, 31, 32, 33, 35, 37, 38,and 41. In some embodiments, the GIP agonist peptides comprise astructure based on a parent sequence comprising any of SEQ ID NOs: 30,31, 32, 33, 35, 37, 38, 41, 298, 299, 300, 301, 303, 305, 306, 309, 531,532, 533, 534, 536, 538, 539, and 542, but differs from the parentsequence at one or more positions, as further described herein. Inexemplary aspects of the peptides comprising a structure based on aparent sequence, comprising any of SEQ ID NOs: 30, 31, 32, 33, 35, 37,38, 41, 298, 299, 300, 301, 303, 305, 306, 309, 531, 532, 533, 534, 536,538, 539, and 542, but differs from the parent sequence at one or morepositions, the amino acid at position 29 is not Gly when the amino acidat position 28 is Asp. In exemplary aspects of the peptides comprising astructure based on a parent sequence, comprising any of SEQ ID NOs: 30,31, 32, 33, 35, 37, 38, 41, 298, 299, 300, 301, 303, 305, 306, 309, 531,532, 533, 534, 536, 538, 539, and 542, but differs from the parentsequence at one or more positions, (i) the amino acid at position 1 isan amino acid comprising a side chain imidazole, (ii) the amino acid atposition 2 is a DPP-IV protective amino acid, optionally, an alpha,alpha-disubstituted amino acid, (iii) the C-terminal amino acid of thepeptide is amidated, and (iv) the peptide comprises at position 40 anamino acid covalently attached to a C12 to C18 acyl or alkyl group, whenthe amino acid at position 10 is Tyr, and optionally, the peptidecomprises Gly at position 41.

The invention accordingly provides a peptide comprising, consistingessentially of, or consisting of the sequence of SEQ ID NO: 18. Alsoprovided is a peptide comprising, consisting essentially of, orconsisting of the sequence of SEQ ID NO: 43.

The invention also provides a peptide comprising, consisting essentiallyof, or consisting of any one of SEQ ID NOs: 100-282; SEQ ID NOs:333-515.

The invention also provides a peptide comprising, consisting essentiallyof, or consisting of any one of SEQ ID NOs: 15-28, 34, 36, 39, 42, 43,44, 52, 53, 63-65, 283-296, 302, 304, 307, 310, 311, 312, 320, 321,330-332, 516-529, 535, 537, 540, 543, 544, 545, 553, 554, and 563-565.

The invention also provides a peptide comprising, consisting essentiallyof, or consisting of any one of SEQ ID NOs: 29-42, 297-310, and 530-543.

Additional peptides comprising one of the amino acid sequences of theaforementioned SEQ ID NO: with up to three (e.g., one, two, three) aminoacid modifications are provided herein. In exemplary aspects, the aminoacid at position 29 is not Gly when the amino acid at position 28 isAsp. In exemplary aspects, (i) the amino acid at position 1 is an aminoacid comprising a side chain imidazole, (ii) the amino acid at position2 is a DPP-IV protective amino acid, optionally, an alpha,alpha-disubstituted amino acid, (iii) the C-terminal amino acid of thepeptide is amidated, and (iv) the peptide comprises at position 40 anamino acid covalently attached to a C12 to C18 acyl or alkyl group, whenthe amino acid at position 10 is Tyr, and optionally, the peptidecomprises Gly at position 41.

Also provided herein are dimers and multimers comprising two or more GIPagonist peptides of the present disclosures. Conjugates comprising a GIPagonist peptide of the present disclosures and a conjugate moiety areadditionally provided herein. In some aspects, the conjugate is a fusionpolypeptide comprising the GIP agonist peptide of the presentdisclosures fused to a heterologous peptide. The present disclosuresalso provides kits comprising the GIP agonist peptides, dimers,multimers, or conjugates of the present disclosures (or a combinationthereof).

Pharmaceutical compositions comprising any of the GIP agonist peptides,dimers, multimers, or conjugates of the present disclosures (or acombination thereof) and a pharmaceutically acceptable carrier, diluent,or excipient are further provided by the present disclosures. Thepharmaceutical compositions are preferably sterile and suitable forparenteral administration. The pharmaceutical compositions arecontemplated for use in methods of treating or preventing diabetes orobesity, or medical conditions associated with diabetes or obesity.Accordingly, in exemplary embodiments the present disclosure provides amethod of reducing weight gain or inducing weight loss, a method oftreating or preventing diabetes or obesity, and a method of inducingtemporary paralysis of the intestinal tract. Further applications of thepeptide analogs and pharmaceutical compositions comprising the same areprovided in the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a graph of the change in body weight (%) as a functionof time of DIO mice subcutaneously injected with a vehicle control, apeptide of SEQ ID NO: 18, a peptide of SEQ ID NO: 29, or a peptide ofSEQ ID NO: 10.

FIG. 2 represents a graph of the total change in body weight (asmeasured on Day 14) of DIO mice subcutaneously injected with a vehiclecontrol, a peptide of SEQ ID NO: 10, a peptide of SEQ ID NO: 18, apeptide of SEQ ID NO: 43, a peptide of SEQ ID NO: 44, a peptide of SEQID NO: 45, a peptide of SEQ ID NO: 46, a peptide of SEQ ID NO: 47, or apeptide of SEQ ID NO: 48.

FIG. 3 represents a graph of the total food intake (as measured on Day14) of DIO mice subcutaneously injected with a vehicle control, apeptide of SEQ ID NO: 10, a peptide of SEQ ID NO: 18, a peptide of SEQID NO: 43, a peptide of SEQ ID NO: 44, a peptide of SEQ ID NO: 45, apeptide of SEQ ID NO: 46, a peptide of SEQ ID NO: 47, or a peptide ofSEQ ID NO: 48.

FIG. 4 represents a graph of the change in blood glucose levels (mg/dL;levels of Day 14-levels of Day 0) of DIO mice subcutaneously injectedwith a vehicle control, a peptide of SEQ ID NO: 10, a peptide of SEQ IDNO: 18, a peptide of SEQ ID NO: 43, a peptide of SEQ ID NO: 44, apeptide of SEQ ID NO: 45, a peptide of SEQ ID NO: 46, a peptide of SEQID NO: 47, or a peptide of SEQ ID NO: 48.

FIG. 5 represents a graph of the change in fat mass (g; levels of Day14-levels of Day 0) of DIO mice subcutaneously injected with a vehiclecontrol, a peptide of SEQ ID NO: 10, a peptide of SEQ ID NO: 18, apeptide of SEQ ID NO: 43, a peptide of SEQ ID NO: 44, a peptide of SEQID NO: 45, a peptide of SEQ ID NO: 46, a peptide of SEQ ID NO: 47, or apeptide of SEQ ID NO: 48.

DETAILED DESCRIPTION

The present disclosures provide GIP agonist peptides (e.g., analogs ofnative human glucagon (SEQ ID NO: 1) (also referred to as “glucagonanalogs”), analogs of any of SEQ ID NOs: 19-22, 24-26, and 28 or any ofSEQ ID NOs: 23, 27, and 29-42) which exhibit agonist activity at the GIPreceptor. As used herein, the term “peptide” encompasses a sequence of 2or more amino acids and typically less than 100, or less than 50 aminoacids. The amino acids can be naturally occurring or coded ornon-naturally occurring or non-coded. Non-naturally occurring aminoacids refer to amino acids that do not naturally occur in vivo butwhich, nevertheless, can be incorporated into the peptide structuresdescribed herein. “Non-coded” as used herein refer to an amino acid thatis not an L-isomer of any of the following 20 amino acids: Ala, Cys,Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, Tyr. The term “GIP agonist peptide” refers to a compoundthat binds to and activates downstream signaling of the GIP receptor.The GIP agonist peptide may have any of the levels of activity at theGIP receptor (e.g., absolute activity level or relative activity level),selectivity for the GIP receptor, or GIP percentage potency, describedherein. See, for example, the section entitled GIP Receptor Activity.However, this term should not be construed as limiting the compound tohaving activity at only the GIP receptor. Rather, the GIP agonistpeptides of the present disclosures may exhibit additional activities atother receptors, as further discussed herein. GIP agonist peptides, forexample, may exhibit agonist activity at the GLP-1 receptor and/orglucagon receptor.

Activity of the Presently Disclosed Peptides GIP Receptor Activity

In some or any embodiments, the peptides of the present disclosuresexhibit an EC50 for GIP receptor activation which is in the nanomolarrange. In exemplary embodiments, the EC50 of the peptide at the GIPreceptor is less than 1000 nM, less than 900 nM, less than 800 nM, lessthan 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, lessthan 300 nM, less than 200 nM. In some embodiments, the EC50 of thepeptide at the GIP receptor is about 100 nM or less, e.g., about 75 nMor less, about 50 nM or less, about 25 nM or less, about 10 nM or less,about 5 nM or less, or about 1 nM or less. In some or any embodiments,the peptide of the present disclosures exhibits an EC50 for GIP receptoractivation which is in the picomolar range. In exemplary embodiments,the EC50 of the GIP agonist peptide at the GIP receptor is less than1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than200 pM. In some embodiments, the EC50 of the peptide at the GIP receptoris about 100 pM or less, e.g., about 75 pM or less, about 50 pM or less,about 25 pM or less, about 10 pM or less, about 5 pM or less, or about 1pM or less. The term “about” as used herein means greater or lesser thanthe value or range of values stated by 10 percent, but is not intendedto designate any value or range of values to only this broaderdefinition. Each value or range of values preceded by the term “about”is also intended to encompass the embodiment of the stated absolutevalue or range of values.

Suitable methods of determining the EC50 of a peptide for activation ofa receptor, e.g., the GIP receptor, are known in the art. One exemplaryin vitro assay, in which cAMP induction as represented by luciferaseactivity is measured in HEK293 cells over-expressing the GIP receptor,is described herein at Example 2.

In some or any embodiments, the peptides of the present disclosuresexhibit enhanced activity at the GIP receptor, as compared to nativehuman glucagon. Native glucagon (SEQ ID NO: 1) does not activate the GIPreceptor; native glucagon exhibits essentially 0% (e.g., less than0.001%, less than 0.0001%) activity of native GIP at the GIP receptor. Apeptide's relative activity at the GIP receptor relative to nativeglucagon is calculated as the inverse ratio of (EC50 of the peptide ofthe present disclosures/EC50 of native glucagon)×100%.

A peptide's relative activity at the GIP receptor compared to native GIPis calculated as the inverse ratio of (EC50 of the peptide of thepresent disclosures/EC50 of native GIP)×100% (a value also referred toherein as “GIP percentage potency”).

In some or any embodiments of the present disclosures, the peptides ofthe present disclosures exhibit GIP percentage potency that is at leastor about 0.1%. In exemplary embodiments, the peptides exhibit at leastor about 0.5%, at least or about 1%, at least or about 5%, at least orabout 10%, at least or about 20%, at least or about 30%, at least orabout 40%, at least or about 50%, at least or about 60%, at least orabout 70%, at least or about 80%, at least or about 90%, or at least orabout 100% of the activity of native GIP at the GIP receptor.

In some embodiments of the present disclosures, the peptides of thepresent disclosures exhibit activity at the GIP receptor which isgreater than that of native GIP. In exemplary embodiments, the GIPagonist peptide exhibits a GIP percentage potency of at least or about125%, at least or about 150%, at least or about 175% at least or about200%, at least or about 300%, at least or about 400%, at least or about500%, at least or about 600%, at least or about 700%, at least or about800%, at least or about 900%, or at least or about 1000%. In someembodiments, the GIP agonist peptides described herein exhibit a GIPpercentage potency of no more than 1000% or no more than 10,000%.

In some aspects, the peptides of the present disclosures exhibit a GIPpercentage potency within the range of about 0.001 to about 10,000percent, or about 0.01 to about 10,000 percent, or about 0.1 to about10,000 percent, or about 1 to about 10,000 percent, or about 0.001 toabout 5000 percent, or about 0.01 to about 5000 percent, or about 0.1 toabout 5000 percent, or about 0.1 to about 1000 percent.

Co-Agonists

In some or any embodiments, the peptide of the present disclosures is aco-agonist peptide insofar as it activates the GIP receptor and a secondreceptor different from the GIP receptor.

GIP/GLP-1 Co-Agonists

By way of example, the peptide of the present disclosures in someaspects exhibits activity at both the GIP receptor and the GLP-1receptor (“GLP-1/GIP receptor co-agonists”). In some aspects, thepeptides exhibit activity at the GLP-1 and GIP receptors, but theglucagon activity has been significantly reduced or destroyed, e.g., byan amino acid modification at position 3. For example, substitution atthis position with an acidic, basic, or a hydrophobic amino acid(glutamic acid, ornithine, norleucine) reduces glucagon activity. Insome or any embodiments, the GIP agonist peptide is a peptide whichexhibits about 10% or less (e.g., about 5% or less, or about 1% or less,or about 0.1% or less) of the activity of native glucagon at theglucagon receptor.

In some or any embodiments, the EC50 of the peptide of the presentdisclosures at the GIP receptor is within about 50-fold or less, about40-fold or less, about 30-fold or less, about 20-fold or less, or about10-fold or less, or about 5-fold or less different (higher or lower)from its EC50 at the GLP-1 receptor. For example, the EC50 at the GIPreceptor can be 10-fold higher or 10-fold lower than the EC50 at theGLP-1 receptor. In some or any embodiments, the GIP percentage potencyof the peptide of the present disclosures is less than or about 50-,40-, 30-, 20-, 10-, or 5-fold different (higher or lower) from its GLP-1percentage potency.

Accordingly, the peptide of the present disclosures has less than100-fold selectivity for the human GLP-1 receptor versus the GIPreceptor. As used herein, the term “selectivity” of a molecule for afirst receptor relative to a second receptor refers to the followingratio: EC50 of the molecule at the second receptor divided by the EC50of the molecule at the first receptor. For example, a molecule that hasan EC50 of 1 nM at a first receptor and an EC50 of 100 nM at a secondreceptor has 100-fold selectivity for the first receptor relative to thesecond receptor. In exemplary embodiments, the selectivity of thepeptide of the present disclosures for the human GLP-1 receptor versusthe GIP receptor is less than 100-fold (e.g., less than or about90-fold, less than or about 80-fold, less than or about 70-fold, lessthan or about 60-fold, less than or about 50-fold, less than or about40-fold, less than or about 30-fold, less than or about 20-fold, lessthan or about 10-fold, less than or about 5-fold).

In some or any embodiments, the peptides of the present disclosuresexhibit enhanced activity at the GLP-1 receptor, as compared to nativehuman glucagon. Native glucagon has about 1% of the activity of nativeGLP-1 at the GLP-1 receptor. A peptide's relative activity at the GLP-1receptor relative to native glucagon is calculated as the inverse ratioof (EC50 of the peptide of the present disclosures/EC50 of nativeglucagon)×100%.

A peptide's relative activity at the GLP-1 receptor compared to nativeGLP-1 is calculated as the inverse ratio of (EC50 of the peptide of thepresent disclosures/EC50 of native GLP-1)×100% (a value referred toherein as “GLP-1 percentage potency”).

In some or any embodiments of the present disclosures, the peptides ofthe present disclosures exhibit a GLP-1 percentage potency of at leastor about 0.1%. In exemplary embodiments, the peptides exhibit a GLP-1percentage potency of at least or about 0.5%, at least or about 1%, atleast or about 5%, at least or about 10%, at least or about 20%, atleast or about 30%, at least or about 40%, at least or about 50%, atleast or about 60%, at least or about 70%, at least or about 80%, atleast or about 90%, or at least or about 100%.

In some embodiments of the present disclosures, the peptides of thepresent disclosures exhibit activity at the GLP-1 receptor which isgreater than that of native GLP-1. In exemplary embodiments, the peptideof the present disclosures exhibits a GLP-1 percentage potency of atleast or about 125%, at least or about 150%, at least or about 175% atleast or about 200%, at least or about 300%, at least or about 400%, atleast or about 500%, at least or about 600%, at least or about 700%, atleast or about 800%, at least or about 900%, or at least or about 1000%.In some embodiments, the peptides of the present disclosures exhibit aGLP-1 percentage potency of no more than 1000% or no more than 10,000%.

GIP/Glucagon Co-Agonists

By way of another example, the peptide of the present disclosures insome aspects exhibits activity at both the GIP receptor and the glucagonreceptor (“glucagon/GIP receptor co-agonists”). In some embodiments,GLP-1 activity has been significantly reduced or destroyed, e.g., by anamino acid modification at position 7, e.g., substitution with Ile, adeletion of the amino acid(s) C-terminal to the amino acid at position27 or 28, yielding a 27- or 28-amino acid peptide, or a combinationthereof. In some embodiments, the peptide of the present disclosures isa peptide which exhibits about 10% or less (e.g., about 5% or less, orabout 1% or less, or about 0.1% or less) of the activity of native GLP-1at the GLP-1 receptor.

In some or any embodiments, the EC50 of the peptide of the presentdisclosures at the GIP receptor is within about 50-fold or less, about40-fold or less, about 30-fold or less, about 20-fold or less, or about10-fold or less, or about 5-fold or less different (higher or lower)from its EC50 at the glucagon receptor. In some or any embodiments, theGIP percentage potency of the peptide of the present disclosures is lessthan or about 50-, 40-, 30-, 20-, 10-, or 5-fold different (higher orlower) from its glucagon percentage potency.

In some embodiments, the peptides of the present disclosures exhibitenhanced activity at the glucagon receptor, as compared to native humanglucagon. A peptide's relative activity at the glucagon receptorcompared to native glucagon is calculated as the inverse ratio of (EC50of the peptide of the present disclosures/EC50 of native glucagon)×100%(a value referred to herein as “glucagon percentage potency”).

In some embodiments of the present disclosures, the peptides of thepresent disclosures exhibit a glucagon percentage potency of at least orabout 0.1%. In exemplary embodiments, the peptides exhibit a glucagonpercentage potency of at least or about 0.5%, at least or about 1%, atleast or about 5%, at least or about 10%, at least or about 20%, atleast or about 30%, at least or about 40%, at least or about 50%, atleast or about 60%, at least or about 70%, at least or about 80%, atleast or about 90%, or at least or about 100%.

In some embodiments of the present disclosures, the peptides of thepresent disclosures exhibit activity at the glucagon receptor which isgreater than that of native glucagon. In exemplary embodiments, thepeptide of the present disclosures exhibits a glucagon percentagepotency of at least or about 125%, at least or about 150%, at least orabout 175% at least or about 200%, at least or about 300%, at least orabout 400%, at least or about 500%, at least or about 600%, at least orabout 700%, at least or about 800%, at least or about 900%, or at leastor about 1000%. In some embodiments, the peptides of the presentdisclosures exhibit a glucagon percentage potency of no more than 1000%or no more than 10,000%.

Triagonists

In some embodiments, the peptides of the present disclosures exhibitactivity at two or more receptors, other than the GIP receptor.Accordingly, the present disclosures provide in some aspects GIPtriagonist peptides. In some aspects, the peptides of the presentdisclosures exhibit activity at each of the glucagon, GIP and GLP-1receptors (“glucagon/GIP/GLP-1 tri-agonists”).

In some embodiments, the EC50 of the peptide of the present disclosuresat the GIP receptor is within 50-fold or less, 20-fold or less, or10-fold or less different (higher or lower) than the EC50 of the peptideof the present disclosures at (a) the GLP-1 receptor, (b) the glucagonreceptor, or both. In some embodiments, the EC50 of the peptide of thepresent disclosures at the GIP receptor is within about 40-fold, about30-fold, about 20-fold different (higher or lower) from its EC50 at theGLP-1 receptor, and optionally within about 50-fold different from itsEC50 at the glucagon receptor. In some embodiments, the GIP percentagepotency of the peptide of the present disclosures is less than or about50-fold, 20-fold or 10-fold different (higher or lower) from (a) itsGLP-1 percentage potency, (b) its glucagon percentage potency, or both.In some embodiments, the GIP percentage potency of the peptide of thepresent disclosures is within about 40-fold, about 30-fold, about20-fold different (higher or lower) from its GLP-1 percentage potency,and optionally within about 50-fold different from its its glucagonpercentage potency. In some embodiments, the peptide of the presentdisclosures does not have at least 100-fold selectivity for the humanGLP-1 receptor versus the GIP receptor. In exemplary embodiments, theselectivity of the peptide of the present disclosures for the humanGLP-1 receptor versus the GIP receptor is less than 100-fold (e.g., lessthan or about 90-fold, less than or about 80-fold, less than or about70-fold, less than or about 60-fold, less than or about 50-fold, lessthan or about 40-fold, less than or about 30-fold, less than or about20-fold, less than or about 10-fold, less than or about 5-fold).

GIP Agonism in the Absence of GLP-1 Agonism and Glucagon Agonism

In some embodiments, the peptide of the present disclosures exhibitsactivity at only the GIP receptor, and not at any other receptor, e.g.,GLP-1 receptor, glucagon receptor. In exemplary embodiments, the peptideof the present disclosures exhibits activity at the GIP receptor, andthe glucagon and GLP-1 activity have been significantly reduced ordestroyed, e.g., by amino acid modifications at positions 3 and 7. Insome embodiments, the peptide of the present disclosures is a peptidewhich exhibits a GLP-1 percentage potency of about 10% or less (e.g.,about 5% or less, or about 1% or less, or about 0.1% or less). In someembodiments, the peptide of the present disclosures is a peptide whichexhibits a glucagon percentage potency of about 10% or less (e.g., about5% or less, or about 1% or less, or about 0.1% or less).

Activity of Conjugates

In some or any embodiments, when the peptide of the present disclosuresis conjugated to a heterologous moiety (e.g., a hydrophilic moiety(e.g., a polyethylene glycol)), as further described herein, the peptideof the present disclosures exhibits a decreased activity (e.g., a lowerpercentage potency or higher EC50) than when the peptide of the presentdisclosures is in a free or unconjugated form. Thus, it is contemplatedthat when any of the foregoing absolute activity levels (e.g. GIPpercentage potency, GLP-1 percentage potency or glucagon percentagepotency, but not relative ratios) is applied to a peptide in conjugatedform, e.g. pegylated, such absolute activity levels are reduced by about10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold, and that suchfold reduced activity levels are contemplated within the scope of thedisclosure. Conversely, when unconjugated, the peptide of the presentdisclosures exhibits a GIP percentage potency that is about 10-fold,about 20-fold, about 30-fold, about 40-fold, about 50-fold, about100-fold or more higher than the potency of the peptide of the presentdisclosures when conjugated to a heterologous moiety (e.g., ahydrophilic moiety (e.g., a polyethylene glycol)).

Structure of the Presently Disclosed Peptides

Glucagon Analogs

In some embodiments, the peptides of the present disclosures arestructurally similar to native human glucagon (SEQ ID NO: 1), e.g., isan analog of native human glucagon (also referred to herein as “glucagonanalog” or “peptide analog of glucagon”). As used herein, the terms“glucagon analog” and “peptide analog of glucagon,” and the like, referto peptides that are structurally similar to native human glucagon andthese terms do not necessarily imply that the peptides activate theglucagon receptor.

In some or any embodiments, the peptide of the present disclosures is ananalog of native human glucagon (SEQ ID NO: 1) comprising an amino acidsequence based on the amino acid sequence of SEQ ID NO: 1 but differsfrom SEQ ID NO: 1 inasmuch as the amino acid sequence of the glucagonanalog comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, and in some instances, 16 or more (e.g., 17, 18, 19, 20,21, 22, 23, 24, 25, etc.), specified or optional amino acidmodifications. In some or any embodiments, the peptide of the presentdisclosures comprises a total of 1, up to 2, up to 3, up to 4, up to 5,up to 6, up to 7, up to 8, up to 9, or up to 10 additional amino acidmodifications (e.g., in addition to the specified amino acidmodifications), relative to the native human glucagon sequence (SEQ IDNO: 1). In some or any embodiments, the modifications are any of thosedescribed herein, e.g., acylation, alkylation, pegylation, truncation atC-terminus, substitution of the amino acid at one or more of positions1, 2, 3, 7, 10, 12, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29.

As used herein an “amino acid modification” refers to (i) a substitutionof an amino acid of SEQ ID NO: 1 with a different amino acid(naturally-occurring or coded or non-coded or non-naturally-occurringamino acid), (ii) an addition of an amino acid (naturally-occurring orcoded or non-coded or non-naturally-occurring amino acid), to SEQ ID NO:1 or (iii) a deletion of one or more amino acids of SEQ ID NO: 1.

In some or any embodiments, the amino acid substitution is aconservative amino acid substitution, e.g., a conservative substitutionof the amino acid at one or more of positions 2, 5, 7, 10, 11, 12, 13,14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29. As used herein, the term“conservative amino acid substitution” is defined herein as thesubstitution of one amino acid with another amino acid having similarproperties, e.g., size, charge, hydrophobicity, hydrophilicity, and/oraromaticity, and includes exchanges within one of the following fivegroups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides and esters:

-   -   Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid;

III. Polar, positively charged residues:

-   -   His, Arg, Lys; Ornithine (Orn)

IV. Large, aliphatic, nonpolar residues:

-   -   Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp, acetyl phenylalanine

In alternative embodiments, the amino acid substitution is not aconservative amino acid substitution, e.g., is a non-conservative aminoacid substitution.

As used herein the term “charged amino acid” refers to an amino acidthat comprises a side chain that is negative-charged (i.e.,de-protonated) or positive-charged (i.e., protonated) in aqueoussolution at physiological pH. For example negative-charged amino acidsinclude aspartic acid, glutamic acid, cysteic acid, homocysteic acid,and homoglutamic acid, whereas positive-charged amino acids includearginine, lysine and histidine. Charged amino acids include the chargedamino acids among the 20 coded amino acids, as well as atypical ornon-naturally occurring or non-coded amino acids. As used herein theterm “acidic amino acid” refers to an amino acid that comprises a secondacidic moiety (other than the alpha carboxylic acid of the amino acid),including for example, a side chain carboxylic acid or sulfonic acidgroup.

In some embodiments, the peptide of the present disclosures comprises anamino acid sequence which has at least 25% sequence identity to theamino acid sequence of native human glucagon (SEQ ID NO: 1). In someembodiments, the peptide of the present disclosures comprises an aminoacid sequence which is at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90% or hasgreater than 90% sequence identity to SEQ ID NO: 1. In some embodiments,the amino acid sequence of the presently disclosed peptide which has theabove-referenced % sequence identity is the full-length amino acidsequence of the presently disclosed peptide. In some embodiments, theamino acid sequence of the peptide of the present disclosures which hasthe above-referenced % sequence identity is only a portion of the aminoacid sequence of the presently disclosed peptide. In some embodiments,the presently disclosed peptide comprises an amino acid sequence whichhas about A % or greater sequence identity to a reference amino acidsequence of at least 5 contiguous amino acids (e.g., at least 6, atleast 7, at least 8, at least 9, at least 10 amino acids) of SEQ ID NO:1, wherein the reference amino acid sequence begins with the amino acidat position C of SEQ ID NO: 1 and ends with the amino acid at position Dof SEQ ID NO: 1, wherein A is 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99; C is 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, or 28 and D is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29. Any and allpossible combinations of the foregoing parameters are envisioned,including but not limited to, e.g., wherein A is 90% and C and D are 1and 27, or 6 and 27, or 8 and 27, or 10 and 27, or 12 and 27, or 16 and27.

The analogs of native human glucagon (SEQ ID NO: 1) described herein maycomprise a peptide backbone of any number of amino acids, i.e., can beof any peptide length. In some embodiments, the peptides describedherein are the same length as SEQ ID NO: 1, i.e., are 29 amino acids inlength. In some embodiments, the presently disclosed peptide is longerthan 29 amino acids in length, e.g., the presently disclosed peptidecomprises a C-terminal extension of 1-21 amino acids, as furtherdescribed herein. Accordingly, the peptide of the present disclosures insome embodiments, is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In someembodiments, the presently disclosed peptide is up to 50 amino acids inlength. In some embodiments, the presently disclosed peptide is longerthan 29 amino acids in length (e.g., greater than 50 amino acids, (e.g.,at least or about 60, at least or about 70, at least or about 80, atleast or about 90, at least or about 100, at least or about 150, atleast or about 200, at least or about 250, at least or about 300, atleast or about 350, at least or about 400, at least or about 450, atleast or about 500 amino acids in length) due to fusion with anotherpeptide. In other embodiments, the presently disclosed peptide is lessthan 29 amino acids in length, e.g., 28, 27, 26, 25, 24, 23, aminoacids.

In accordance with the foregoing, in some aspects, the peptide of thepresent disclosures is an analog of native human glucagon (SEQ ID NO: 1)comprising an amino acid sequence based on SEQ ID NO: 1, which sequencecomprises one or more amino acid modifications which affect GIPactivity, glucagon activity, and/or GLP-1 activity, enhance stability,e.g., by reducing degradation of the peptide (e.g., by improvingresistance to DPP-IV proteases), enhance solubility, increase half-life,delay the onset of action, extend the duration of action at the GIP,glucagon, or GLP-1 receptor, or a combination of any of the foregoing.Such amino acid modifications, in addition to other modifications, arefurther described below, and any of these modifications can be appliedindividually or in combination.

Amino Acids Comprising a Non-Native Acyl Group

In accordance with some or any embodiments, the GIP agonist peptideswhich are analogs of glucagon (SEQ ID NO: 1) comprise an amino acidcomprising a non-native acyl group (referred to herein as an “acylatedamino acid”, regardless of how it is prepared, e.g., by incorporation ofa previously-acylated amino acid into the peptide, or acylation of thepeptide after synthesis). In some or any aspects, the acylated aminoacid is located at any of positions 9, 10, 12, 13, 14, 16, 17, 20, 37,38, 39, 40, 41, 42, or 43 of the glucagon analog. In exemplary aspects,the acylated amino acid is located at any of positions 9, 10, 12, 16,20, or 40 of the glucagon analog or at any of positions 10, 13, 14, 16,17, or 40 of the glucagon analog. In exemplary aspects, the acylatedamino acid is located at any one or more of positions 10, 14, and 40. Inexemplary aspects, the acylated amino acid is located at any ofpositions 10, 12, or 16 of the peptide analog.

The acylated amino acid in some embodiments causes the GIP agonistpeptide to have one or more of (i) a prolonged half-life in circulation,(ii) a delayed onset of action, (iii) an extended duration of action,(iv) an improved resistance to proteases, such as DPP-IV, and (v)increased potency at any one or more of the GIP receptor, GLP-1receptor, and glucagon receptor.

Direct Acylation

In some embodiments, the acyl group is directly linked to an amino acidof the GIP agonist peptide. In accordance with one embodiment, the GIPagonist peptide comprises an acyl group which is attached to the peptidevia an ester, thioester, or amide linkage.

In specific aspects, the GIP agonist peptide comprises an acyl groupupon direct acylation of an amine, hydroxyl, or thiol of a side chain ofan amino acid of the GIP agonist peptide. In some embodiments, acylationis at position 9, 10, 12, 13, 14, 16, 17, 20, or 40 (e.g., at any one ofpositions 10, 14, and 40) of the GIP agonist peptide. In this regard,the GIP agonist peptide comprises the amino acid sequence of SEQ ID NO:1, or a modified amino acid sequence thereof comprising one or more ofthe amino acid modifications described herein, wherein at least one ofthe amino acids at positions 9, 10, 12, 13, 14, 16, 17, 20, and 40(e.g., at any one of positions 10, 14, and 40) of the GIP agonistpeptide is an amino acid comprising a side chain amine, hydroxyl, orthiol.

In some embodiments, the amino acid comprising a side chain amine is anamino acid of Formula I:

In some embodiments, the amino acid of Formula I, is the amino acidwherein n is 4 (Lys) or n is 3 (Orn). In some embodiments, the aminoacid comprising a side chain amine is an aromatic amino acid comprisinga side chain amine. In exemplary aspects, the aromatic amino acidcomprising a side chain amine is 4-amino-phenylalanine (4-aminoPhe),p-amino phenylglycine, p-amino homophenylalanine, or 3-amino tyrosine.In exemplary aspects, the aromatic amino acid comprising a side chainamine is 4-amino-Phe.

In other embodiments, the amino acid comprising a side chain hydroxyl isan amino acid of Formula II:

In some exemplary embodiments, the amino acid of Formula II is the aminoacid wherein n is 1 (Ser). In exemplary aspects, the amino acid ofFormula II is the amino acid wherein n is 2 (homoserine). In similarexemplary embodiments, the amino acid comprising a side chain hydroxylis a Thr or homothreonine. In similar exemplary embodiments, the aminoacid comprising a side chain hydroxyl is an aromatic amino acidcomprising a side chain hydroxyl. In exemplary aspects, the aromaticamino acid comprising a side chain hydroxyl is tyrosine, homotyrosine,methyl-tyrosine, or 3-amino tyrosine.

In yet other embodiments, the amino acid comprising a side chain thiolis an amino acid of Formula III:

In some exemplary embodiments, the amino acid of Formula III is theamino acid wherein n is 1 (Cys).

In yet other embodiments, the amino acid comprising a side chain amine,hydroxyl, or thiol is a disubstituted amino acid comprising the samestructure of Formula I, Formula II, or Formula III, except that thehydrogen bonded to the alpha carbon of the amino acid of Formula I,Formula II, or Formula III is replaced with a second side chain.

Acylation Spacers

In alternative embodiments, the acyl group is linked via a spacer to anamino acid of the GIP agonist peptide, wherein the spacer is positionedbetween the amino acid of the GIP agonist peptide and the acyl group. Insome embodiments, the GIP agonist peptide comprises a spacer between thepeptide and the acyl group. In some embodiments, the GIP agonist peptideis covalently bound to the spacer, which is covalently bound to the acylgroup.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol, or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol. The aminoacid to which the spacer is attached can be any amino acid (e.g., asingly or doubly α-substituted amino acid) comprising a moiety whichpermits linkage to the spacer. For example, an amino acid comprising aside chain NH₂, —OH, or —COOH (e.g., Lys, Orn, Ser, Asp, or Glu) issuitable. In this respect, the GIP agonist peptide in some aspectscomprises the amino acid sequence of SEQ ID NO: 1, or a modified aminoacid sequence thereof comprising one or more of the amino acidmodifications described herein, wherein at least one of the amino acidsat positions 9, 10, 12, 13, 14, 16, 17, 20, and 37-43 (e.g., position10, 14, or 40) is an amino acid comprising a side chain amine, hydroxyl,or carboxylate.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol, or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol.

When the acyl group is attached through an amine group of a spacer, theacyl group in some aspects is attached through the alpha amine orthrough a side chain amine of the spacer amino acid. In the instance inwhich the acyl group is attached via an alpha amine, the amino acid ofthe spacer can be any amino acid. For example, the amino acid of thespacer can be a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu, Ile,Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5-aminovaleric acid,7-aminoheptanoic acid, and 8-aminooctanoic acid. Alternatively, in someaspects, the amino acid of the spacer is an acidic residue, e.g., Asp,Glu, homoglutamic acid, homocysteic acid, cysteic acid, gamma-glutamicacid.

In the instance in which the acyl group is attached through a side chainamine of the amino acid spacer, the spacer is an amino acid comprising aside chain amine, e.g., an amino acid of Formula I (e.g., Lys or Orn).In this instance, it is possible for both the alpha amine and the sidechain amine of the amino acid of the spacer to be attached to an acylgroup, such that the GIP agonist peptide is diacylated. Embodiments ofthe invention include such diacylated molecules. In some embodiments,the acyl group is attached to a 4-amino-Phe, p-amino phenylglycine,p-amino homophenylalanine, or 3-amino tyrosine.

When the acyl group is attached through a hydroxyl group of a spacer,the amino acid or one of the amino acids of the dipeptide or tripeptidecan be an amino acid of Formula II. In a specific exemplary embodiment,the amino acid is Ser. In similar exemplary embodiments, the acyl groupis attached to a Thr or homothreonine. In similar exemplary embodiments,the acyl group is attached via the hydroxyl of an aromatic amino acidcomprising a side chain hydroxyl, e.g., tyrosine, homotyrosine,methyl-tyrosine, or 3-amino tyrosine.

When the acyl group is attached through a thiol group of a spacer, theamino acid or one of the amino acids of the dipeptide or tripeptide canbe an amino acid of Formula III. In a specific exemplary embodiment, theamino acid is Cys.

In some embodiments, the spacer is a hydrophilic bifunctional spacer. Inexemplary embodiments, the hydrophilic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In exemplary embodiments,the hydrophilic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydrophilic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydrophilic bifunctional spacer comprises a thiol group and acarboxylate. In a specific embodiment, the spacer comprises an aminopoly(alkyloxy)carboxylate. In this regard, the spacer can comprise, forexample, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.).

In exemplary embodiments, the spacer comprises a small polyethyleneglycol moiety (PEG) comprising a structure [—O—CH₂—CH₂—]_(n), wherein nis an integer between 2 and 16, (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16). Such small PEGs are referred to herein as a“miniPEG.” In exemplary aspects, the miniPEG is a functionalized miniPEGcomprising one or more functional groups. Suitable functional groupsinclude, but are not limited to, an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In exemplary aspects, theminiPEG is a miniPEG acid comprising a structure{[—O—CH₂—CH₂—]_(n)—COO—}, wherein n is defined as above. In exemplaryaspects, the miniPEG is an amido miniPEG comprising a structure{—N—CH₂—CH₂—[—O—CH₂—CH₂—]_(n)}, wherein n is defined as above. Inexemplary aspects, the miniPEG is an amido miniPEG acid comprising astructure {—N—CH₂—CH₂—[—O—CH₂—CH₂—]_(n)—COO—}, wherein n is defined asabove. Suitable reagents for use in acylating an amino acid with aminiPEG are commercially available from vendors, such as PeptidesInternational (Louisville, Ky.). Also, suitable techniques for acylatingan amino acid with a miniPEG are described herein (see Example 1).

In some embodiments, the spacer is a hydrophobic bifunctional spacer.Hydrophobic bifunctional spacers are known in the art. See, e.g.,Bioconjugate Techniques, G. T. Hermanson (Academic Press, San Diego,Calif., 1996), which is incorporated by reference in its entirety. Inexemplary embodiments, the hydrophobic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In exemplary embodiments,the hydrophobic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydrophobic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydrophobic bifunctional spacer comprises a thiol group and acarboxylate. Suitable hydrophobic bifunctional spacers comprising acarboxylate and a hydroxyl group or a thiol group are known in the artand include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoicacid.

In some embodiments, the bifunctional spacer is not a dicarboxylic acidcomprising an unbranched, methylene of 1-7 carbon atoms between thecarboxylate groups. In some embodiments, the bifunctional spacer is adicarboxylic acid comprising an unbranched, methylene of 1-7 carbonatoms between the carboxylate groups.

The spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilicbifunctional spacer, or hydrophobic bifunctional spacer) in specificembodiments is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or10 atoms) in length. In more specific embodiments, the spacer is about 3to 10 atoms (e.g., 6 to 10 atoms) in length and the acyl group is a C12to C18 fatty acyl group, e.g., C14 fatty acyl group, C16 fatty acylgroup, such that the total length of the spacer and acyl group is 14 to28 atoms, e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, or 28 atoms. In some embodiments, the length of the spacer andacyl group is 17 to 28 (e.g., 19 to 26, 19 to 21) atoms.

In accordance with some or any of the foregoing embodiments, thebifunctional spacer can be a synthetic or naturally occurring amino acid(including, but not limited to, any of those described herein)comprising an amino acid backbone that is 3 to 10 atoms in length (e.g.,6-amino hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoic acid, and8-aminooctanoic acid). Alternatively, the spacer can be a dipeptide ortripeptide spacer having a peptide backbone that is 3 to 10 atoms (e.g.,6 to 10 atoms) in length. Each amino acid of the dipeptide or tripeptidespacer can be the same as or different from the other amino acid(s) ofthe dipeptide or tripeptide and can be independently selected from thegroup consisting of: naturally-occurring or coded and/or non-coded ornon-naturally occurring amino acids, including, for example, any of theD or L isomers of the naturally-occurring amino acids (Ala, Cys, Asp,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val,Trp, Tyr), or any D or L isomers of the non-naturally occurring ornon-coded amino acids selected from the group consisting of: β-alanine(β-Ala), N-α-methyl-alanine (Me-Ala), aminobutyric acid (Abu),γ-aminobutyric acid (γ-Abu), aminohexanoic acid (ε-Ahx), aminoisobutyricacid (Aib), aminomethylpyrrole carboxylic acid,aminopiperidinecarboxylic acid, aminoserine (Ams),aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methylamide, β-aspartic acid (β-Asp), azetidine carboxylic acid,3-(2-benzothiazolyl)alanine, α-tert-butylglycine,2-amino-5-ureido-n-valeric acid (citrulline, Cit), β-Cyclohexylalanine(Cha), acetamidomethyl-cysteine, diaminobutanoic acid (Dab),diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA),dimethylthiazolidine (DMTA), γ-Glutamic acid (γ-Glu), homoserine (Hse),hydroxyproline (Hyp), isoleucine N-methoxy-N-methyl amide,methyl-isoleucine (MeIle), isonipecotic acid (Isn), methyl-leucine(MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine,methanoproline, methionine-sulfoxide (Met(O)), methionine-sulfone(Met(O)₂), norleucine (Nle), methyl-norleucine (Me-Nle), norvaline(Nva), ornithine (Orn), para-aminobenzoic acid (PABA), penicillamine(Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4-C1)),4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-NO₂)),4-cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg),piperidinylalanine, piperidinylglycine, 3,4-dehydroproline,pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec),O-Benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta),4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA),4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA),1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid (Tic),tetrahydropyranglycine, thienylalanine (Thi), O-benzyl-phosphotyrosine,O-Phosphotyrosine, methoxytyrosine, ethoxytyrosine,O-(bis-dimethylamino-phosphono)-tyrosine, tyrosine sulfatetetrabutylamine, methyl-valine (MeVal), and alkylated3-mercaptopropionic acid. In exemplary aspects, the spacer is a Cysresidue or a Lys residue.

In some embodiments, the spacer comprises an overall negative charge,e.g., comprises one or two negative-charged amino acids. In someembodiments, the dipeptide is not any of the dipeptides of generalstructure A-B, wherein A is selected from the group consisting of Gly,Gln, Ala, Arg, Asp, Asn, Ile, Leu, Val, Phe, and Pro, wherein B isselected from the group consisting of Lys, His, Trp. In someembodiments, the amino acids of the dipeptide spacer are selected fromthe group consisting of: Ala, β-Ala, Leu, Pro, γ-aminobutyric acid, Gluand γ-Glu.

In some exemplary embodiments, the GIP agonist peptide comprises an acylgroup upon acylation of an amine, hydroxyl, or thiol of a spacer, whichspacer is attached to a side chain of an amino acid at position 9, 10,12, 13, 14, 16, 17, 20, or 37-43 (e.g., at any one or more of positions10, 14, and 40), or at the C-terminal amino acid of the GIP agonistpeptide.

In yet more specific embodiments, the acyl group is attached to theamino acid at any of positions 9, 10, 12, 13, 14, 16, 17, 20, or 37-43(e.g., at any one or more of positions 10, 14, and 40) of the peptideanalog and the length of the spacer and acyl group is 14 to 28 atoms.The amino acid at any of positions 9, 10, 12, 13, 14, 16, 17, 20, or37-43 (e.g., at any one or more of positions 10, 14, and 40), in someaspects, is an amino acid of Formula I, e.g., Lys, or a disubstitutedamino acid related to Formula I. In more specific embodiments, thepeptide analog lacks an intramolecular bridge, e.g., a covalentintramolecular bridge. The glucagon analog, for example, can be aglucagon analog comprising one or more alpha, alpha-disubstituted aminoacids, e.g., AIB, for stabilizing the alpha helix of the analog.

Acyl Groups

The acyl group of the acylated amino acid can be of any size, e.g., anylength carbon chain, and can be linear or branched. In some specificembodiments, the acyl group is a C4 to C30 fatty acid. For example, theacyl group can be any of a C4 fatty acid, C6 fatty acid, C8 fatty acid,C10 fatty acid, C12 fatty acid, C14 fatty acid, C16 fatty acid, C18fatty acid, C20 fatty acid, C22 fatty acid, C24 fatty acid, C26 fattyacid, C28 fatty acid, or a C30 fatty acid. In some embodiments, the acylgroup is a C8 to C20 fatty acid, e.g., a C12 to C18 fatty acid, a C14fatty acid or a C16 fatty acid. In exemplary embodiments, the acyl groupis a C16 to C30 fatty acid, e.g., a C20 to C28 fatty acid, a C16 to C22fatty acid.

In an alternative embodiment, the acyl group is a bile acid. The bileacid can be any suitable bile acid, including, but not limited to,cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid,taurocholic acid, glycocholic acid, and cholesterol acid.

In exemplary embodiments, the acyl group is a succinic acid or asuccinic acid derivative. By “succinic acid derivative” as used hereinis meant a compound comprising a substituted succinic acid or asubstituted cyclic succinic acid (i.e., succinic anhydride) or asubstituted expanded ring succinic anhydride, (i.e. a 6-8 membered ringcomprising the —C(O)—O—C(O)— moiety and 3 to 5 additional carbons),wherein the substituted succinic acid, substituted cyclic succinic acid(i.e., succinic anhydride), or substituted expanded ring succinicanhydride is substituted with one or more alkyl chains or one or morefunctionalized carbon chains.

In exemplary aspects, the succinic acid derivative comprises a structureof Formula V:

wherein each of R and R′ is independently H, a linear or branched C4-C30carbon chain, or a linear or branched C4-C30 functionalized carbonchain. In exemplary embodiments, R and/or R′ is a carbon chaincomprising a C4 to C30 alkyl chain. For example, the alkyl group can beany of a C4 alkyl, C6 alkyl, C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl,C16 alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl, C26 alkyl, C28alkyl, or a C30 alkyl. In some embodiments, the alkyl group is a C8 toC20 alkyl, e.g., a C12 to C18 alkyl, a C14 alkyl or a C16 alkyl. Inexemplary embodiments, the alkyl group is a C16 to C30 alkyl, e.g., aC20 to C28 alkyl, a C16 to C22 alkyl. In exemplary aspects, thefunctionalized carbon chain comprises a functional group, including, butnot limited, carboxyl, sulfhydryl, amine, ketyl, sulfoxyl or amido.

In exemplary aspects, the succinic acid derivative comprises a succinicanhydride comprising a structure of Formula VI:

wherein each of R and R′ is independently H, a linear or branched C4-C30carbon chain, or a linear or branched C4-C30 functionalized carbonchain. In exemplary embodiments, R and/or R′ is a carbon chaincomprising a C4 to C30 alkyl chain. For example, the alkyl group can beany of a C4 alkyl, C6 alkyl, C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl,C16 alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl, C26 alkyl, C28alkyl, or a C30 alkyl. In some embodiments, the alkyl group is a C8 toC20 alkyl, e.g., a C12 to C18 alkyl, a C14 alkyl or a C16 alkyl. Inexemplary embodiments, the alkyl group is a C16 to C30 alkyl, e.g., aC20 to C28 alkyl, a C16 to C22 alkyl. In exemplary aspects, thefunctionalized carbon chain comprises a functional group, including, butnot limited, carboxyl, sulfhydryl, amine, ketyl, sulfoxyl or amido.

In exemplary aspects, the succinic acid derivative is a succinicanhydride derivative, including those of Formula VII:

-   -   wherein n is 1-4, and        wherein each of R and R′ is independently H, a linear or        branched C4-C30 carbon chain, or a linear or branched C4-C30        functionalized carbon chain. In exemplary embodiments, R and/or        R′ is a carbon chain comprising a C4 to C30 alkyl chain. For        example, the alkyl group can be any of a C4 alkyl, C6 alkyl, C8        alkyl, C10 alkyl, C12 alkyl, C14 alkyl, C16 alkyl, C18 alkyl,        C20 alkyl, C22 alkyl, C24 alkyl, C26 alkyl, C28 alkyl, or a C30        alkyl. In some embodiments, the alkyl group is a C8 to C20        alkyl, e.g., a C12 to C18 alkyl, a C14 alkyl or a C16 alkyl. In        exemplary embodiments, the alkyl group is a C16 to C30 alkyl,        e.g., a C20 to C28 alkyl, a C16 to C22 alkyl. In exemplary        aspects, the functionalized carbon chain comprises a functional        group, including, but not limited, carboxyl, sulfhydryl, amine,        ketyl, sulfoxyl or amido.

When only one of R and R′ of Formulae V-VI is H, the acylated amino acidis referred to as “Cx Succinoyl.” As used herein, the term “CxSuccinoyl,” wherein x is an integer, refers to a structure wherein R isan alkyl chain of y carbons and y=x−1, and y does not include thecarbons of succinoyl moiety. For example a structure of Formula VIwherein R is a C15 alkyl group and R′ is a H is referred to as C16Succinoyl. For example, FIG. 3A depicts a C16 Succinoyl Lys, wherein aLys has been succinoylated at the ε amine. When neither R nor R′ ofFormulae V-VI is H, then the acylated amino acid is referred to as “Cx,Cx′Succinoyl.” As used herein, the term “Cx, Cx′Succinoyl,” wherein xand x′ are integers, refers to a structure wherein R is an alkyl chainof y carbons and R′ is an alkyl chain of y′ carbons, and y′=x′−1. Forexample, a structure of Formula VI wherein R is a C15 alkyl group and R′is a C13 alkyl group is referred to as C16,C14 succinoyl. When thesuccinic acid derivative is a substituted expanded ring succinicanhydride and neither R nor R′ of Formula VII is H, then the acylatedamino acid is referred to as “Cx, Cx′-n-Succinoyl.” As used herein, theterm “Cx,Cx′-n-Succinoyl,” wherein x, x′, and n are integers, refers toa structure wherein R is an alkyl chain of y carbons, R′ is an alkylchain of y′ carbons, and the succinic anhydride ring is extend by ncarbons. For example, a structure of Formula VII wherein R and R′ areC15 alkyl groups and n=2 is referred to as C16,C16-2-Succinoyl.

In exemplary embodiments, the acyl group is a maleic acid or a maleicacid derivative. By “maleic acid derivative” as used herein is meant acompound comprising a substituted maleic acid or a substituted cyclicmaleic acid (i.e., maleic anhydride) or a substituted expanded ringmaleic anhydride, (i.e. a 6-8 membered ring comprising the —C(O)—O—C(O)—moiety and 3 to 5 additional carbons), wherein the substituted maleicacid, substituted cyclic maleic acid (i.e., maleic anhydride), orsubstituted expanded ring maleic anhydride is substituted with one ormore alkyl chains or one or more functionalized carbon chains.

In exemplary aspects, the maleic acid derivative comprises a structureof Formula VIII:

wherein each of R and R′ is independently H, a linear or branched C4-C30carbon chain, or a linear or branched C4-C30 functionalized carbonchain. In exemplary embodiments, R and/or R′ is a carbon chaincomprising a C4 to C30 alkyl chain. For example, the alkyl group can beany of a C4 alkyl, C6 alkyl, C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl,C16 alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl, C26 alkyl, C28alkyl, or a C30 alkyl. In some embodiments, the alkyl group is a C8 toC20 alkyl, e.g., a C12 to C18 alkyl, a C14 alkyl or a C16 alkyl. Inexemplary embodiments, the alkyl group is a C16 to C30 alkyl, e.g., aC20 to C28 alkyl, a C16 to C22 alkyl. In exemplary aspects, thefunctionalized carbon chain comprises a functional group, including, butnot limited, carboxyl, sulfhydryl, amine, ketyl, sulfoxyl or amido.

In exemplary aspects, the maleic acid derivative comprises a maleicanhydride comprising a structure of Formula IX:

wherein each of R and R′ is independently H, a linear or branched C4-C30carbon chain, or a linear or branched C4-C30 functionalized carbonchain. In exemplary embodiments, R and/or R′ is a carbon chaincomprising a C4 to C30 alkyl chain. For example, the alkyl group can beany of a C4 alkyl, C6 alkyl, C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl,C16 alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl, C26 alkyl, C28alkyl, or a C30 alkyl. In some embodiments, the alkyl group is a C8 toC20 alkyl, e.g., a C12 to C18, alkyl, a C14 alkyl or a C16 alkyl. Inexemplary embodiments, the alkyl group is a C16 to C30 alkyl, e.g., aC20 to C28 alkyl, a C16 to C22 alkyl. In exemplary aspects, thefunctionalized carbon chain comprises a functional group, including, butnot limited, carboxyl, sulfhydryl, amine, ketyl, sulfoxyl or amido.

In exemplary aspects, the maleic acid derivative is a maleic anhydridederivative, including those of Formula X:

wherein n is 1-4, there is at least one C═C double bond between twonon-carbonyl carbons, and wherein each of R and R′ is independently H, alinear or branched C4-C30 carbon chain, or a linear or branched C4-C30functionalized carbon chain. In exemplary embodiments, R and/or R′ is acarbon chain comprising a C4 to C30 alkyl chain. For example, the alkylgroup can be any of a C4 alkyl, C6 alkyl, C8 alkyl, C10 alkyl, C12alkyl, C14 alkyl, C16 alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl,C26 alkyl, C28 alkyl, or a C30 alkyl. In some embodiments, the alkylgroup is a C8 to C20 alkyl, e.g., a C12 to C18, alkyl, a C14 alkyl or aC16 alkyl. In exemplary embodiments, the alkyl group is a C16 to C30alkyl, e.g., a C20 to C28 alkyl, a C16 to C22 alkyl. In exemplaryaspects, the functionalized carbon chain comprises a functional group,including, but not limited, carboxyl, sulfhydryl, amine, ketyl, sulfoxylor amido.

When only one of R and R′ of Formulae VIII-IX is H, the acylated aminoacid is referred to as “Cx Maleoyl.” As used herein, the term “CxMaleoyl,” wherein x is an integer, refers to a structure wherein R is analkyl chain of y carbons and y=x−1, and y does not include the carbonsof maleoyl moiety. For example a structure of Formula IX wherein R is aC15 alkyl group and R′ is a H is referred to as C16 Maleoyl. Whenneither R nor R′ of Formulae VIII-IX is H, then the acylated amino acidis referred to as “Cx,Cx′Maleoyl.” As used herein, the term “Cx,Cx′Maleoyl,” wherein x and x′ are integers, refers to a structurewherein R is an alkyl chain of y carbons and R′ is an alkyl chain of y′carbons and y′=x′−1. For example, a structure of Formula IX wherein R isa C15 alkyl group and R′ is a C13 alkyl group is referred to as C16,C14maleoyl. When the maleic acid derivative is a substituted expanded ringmaleic anhydride and neither R nor R′ of Formula X is H, then theacylated amino acid is referred to as “Cx,Cx′-n-Maleoyl.” As usedherein, the term “Cx,Cx′-n-Maleoyl,” wherein x, x′, and n are integers,refers to a structure wherein R is an alkyl chain of y carbons, R′ is analkyl chain of y′ carbons, and the maleic anhydride ring is extend by ncarbons. For example, a structure of Formula X wherein R and R′ are C15alkyl groups and n=2 is referred to as C16,C16-2-Maleoyl.

Methods of Attaching an Acyl Group

Suitable methods of attaching acyl groups to peptides via amines,hydroxyls, and thiols of the peptides are known in the art. See, forexample, Example 1 (for methods of acylating through an amine), Miller,Biochem Biophys Res Commun 218: 377-382 (1996); Shimohigashi andStammer, Int J Pept Protein Res 19: 54-62 (1982); and Previero et al.,Biochim Biophys Acta 263: 7-13 (1972) (for methods of acylating througha hydroxyl); and San and Silvius, J Pept Res 66: 169-180 (2005) (formethods of acylating through a thiol); Bioconjugate Chem. “ChemicalModifications of Proteins: History and Applications” pages 1, 2-12(1990); Hashimoto et al., Pharmacuetical Res. “Synthesis of PalmitoylDerivatives of Insulin and their Biological Activity” Vol. 6, No: 2 pp.171-176 (1989).

In some embodiments, the GIP agonist peptide comprises an acylated aminoacid by acylation of a long chain alkane by the GIP agonist peptide. Inspecific aspects, the long chain alkane comprises an amine, hydroxyl, orthiol group (e.g., octadecylamine, tetradecanol, and hexadecanethiol)which reacts with a carboxyl group, or activated form thereof, of theGIP agonist peptide. The carboxyl group, or activated form thereof, ofthe GIP agonist peptide can be part of a side chain of an amino acid(e.g., glutamic acid, aspartic acid) of the GIP agonist peptide or canbe part of the peptide backbone.

In exemplary embodiments, the GIP agonist peptide comprises an acylgroup by acylation of the long chain alkane by a spacer which isattached to the GIP agonist peptide. In specific aspects, the long chainalkane comprises an amine, hydroxyl, or thiol group which reacts with acarboxyl group, or activated form thereof, of the spacer. Suitablespacers comprising a carboxyl group, or activated form thereof, aredescribed herein and include, for example, bifunctional spacers, e.g.,amino acids, dipeptides, tripeptides, hydrophilic bifunctional spacersand hydrophobic bifunctional spacers.

As used herein, the term “activated form of a carboxyl group” refers toa carboxyl group with the general formula R(C═O)X, wherein X is aleaving group and R is the glucagon analog or the spacer. For example,activated forms of a carboxyl groups may include, but are not limitedto, acyl chlorides, anhydrides, and esters. In some embodiments, theactivated carboxyl group is an ester with a N-hydroxysuccinimide ester(NHS) leaving group.

With regard to these aspects, in which a long chain alkane is acylatedby the glucagon analog or the spacer, the long chain alkane may be ofany size and can comprise any length of carbon chain. The long chainalkane can be linear or branched. In exemplary aspects, the long chainalkane is a C4 to C30 alkane. For example, the long chain alkane can beany of a C4 alkane, C6 alkane, C8 alkane, C10 alkane, C12 alkane, C14alkane, C16 alkane, C18 alkane, C20 alkane, C22 alkane, C24 alkane, C26alkane, C28 alkane, or a C30 alkane. In some embodiments, the long chainalkane comprises a C8 to C20 alkane, e.g., a C12 to C18, alkane, a C14alkane, C16 alkane, or a C18 alkane. In exemplary embodiments, the longchain alkane is a C16 to C30 alkane, e.g., a C20 to C28 alkane, a C16 toC22 alkane.

Also, in some embodiments, an amine, hydroxyl, or thiol group of the GIPagonist peptide is acylated with a cholesterol acid. In a specificembodiment, the GIP agonist peptide is linked to the cholesterol acidthrough an alkylated des-amino Cys spacer, i.e., an alkylated3-mercaptopropionic acid spacer.

When the acyl group is a succinic acid, succinic acid derivative, maleicacid, or maleic acid derivative the peptide is succinoylated/maleoylatedby the reaction of an amine, hydroxyl, or thiol group of the GIP agonistpeptide, or spacer, with a succinic acid, succinic acid derivative,maleic acid, or maleic acid derivative of Formula V, Formula VI, FormulaVII, Formula VIII, Formula IX or formula X. Methods of succinoylationare described herein.

Additional Acyl Groups

The peptide in some or any embodiments comprises an acylated amino acidat a position other than positions 9, 10, 12, 13, 14, 16, 17, 20, or37-43 (e.g., at any one or more of positions 10, 14, and 40). Thelocation of the acylated amino acid may be any position within the GIPagonist peptide, including any of positions 1-29, a position C-terminalto the 29^(th) amino acid (e.g., the amino acid at position 30, 31, 32,33, 34, 35, 36, 44, 45, 46, 47, etc., at a position within a C-terminalextension or at the C-terminus), optionally together with a secondacylated amino acid at any of positions 9, 10, 12, 13, 14, 16, 17, 20,or 37-43 (e.g., at any one or more of positions 10, 14, and 40),provided that the GIP agonist activity of the peptide analog isretained, if not enhanced. Nonlimiting examples include positions 5, 7,11, 13, 14, 17, 18, 19, 21, 24, 27, 28, or 29.

Consistent with the foregoing, the glucagon analog, in exemplaryaspects, comprises two (or more) acylated amino acids, and may beconsidered a dual acylated peptide or a diacylated peptide, when thereare two acyl groups. In exemplary aspects, all of the acylated aminoacids are located at two positions of positions 9, 10, 12, 13, 14, 16,17, 20, or 37-43 (e.g., at any one or more of positions 10, 14, and 40).In exemplary aspects, the acylations occur at two of positions 10, 14,and 40. In exemplary aspects, the peptide comprising a first acylatedamino acid at position 10 and a second acylated amino acid at position40.

In yet additional exemplary embodiments, the glucagon analog comprisestwo (or more) acyl groups attached to a single amino acid of the peptidebackbone. The peptide may be considered as a dual acylated peptide or adiacylated peptide, when there are two acyl groups. The two (or more)acyl groups may be the same acyl group or different acyl groups,arranged in a branched or linear formation. For example, to achieve abranched formation, the glucagon analog may comprise one acylated aminoacid (which is part of the peptide backbone) attached to a spacercomprising at least three functional groups—at least two of which areeach covalently attached to an acyl group and one of which is attachedto the acylated amino acid of the peptide backbone. In exemplaryaspects, a branched formation may be achieved through, e.g., a Lysresidue comprising two amine groups (a side chain amine and an alphaamine) for direct attachment to a fatty acyl group or indirectattachment to a fatty acyl group via a spacer. In exemplary aspects, anadditional spacer may be placed between the amino acid of the peptidebackbone and the spacer comprising at least three functional groups. Forexample, the amino acid of the peptide backbone may be attached (e.g.,via its side chain) to a first spacer, which, in turn, is attached to asecond spacer, wherein the second spacer comprises at least threefunctional groups—at least two of which are each covalently attached toan alkyl group and one of which is attached to the first spacer.

In exemplary aspects, wherein the alkyl groups are arranged in a linearformation, the glucagon analog comprises one acylated amino acid (whichis part of the peptide backbone) directly attached to a first acylgroup, which, in turn, is attached to a spacer, which, in turn, isattached to a second acyl group.

Exemplary structures of dual acylated compounds are depicted in FIG. 5A.

Hydrophilic Moieties and Acyl Groups

The GIP agonist peptides comprising an acylated amino acid optionallyfurther comprises a hydrophilic moiety. In some or any embodiments thehydrophilic moiety comprises a polyethylene glycol (PEG) chain. Theincorporation of a hydrophilic moiety is accomplished through anysuitable means, such as any of the methods described herein. In thisregard, the GIP agonist peptide can comprise SEQ ID NO: 1, including anyof the modifications described herein, in which at least one of theamino acids at any of positions 9, 10, 12, 13, 14, 16, 17 20, and 37-43(e.g., at one or more of positions 10, 14, and 40) is an acylated aminoacid and at least one of the amino acids at position 16, 17, 21, 24, or29, a position within a C-terminal extension, or the C-terminal aminoacid is a Cys, Lys, Orn, homo-Cys, or Ac-Phe, of which the side chain iscovalently bonded to a hydrophilic moiety (e.g., PEG). In someembodiments, the amino acid at any of positions 9, 10, 12, 13, 14, 16,17, 20, and 37-43 (e.g., at one or more of positions 10, 14, and 40) ofthe GIP agonist peptide is attached (optionally via a spacer) to theacyl group and that amino acid is further attached to a hydrophilicmoiety or is further attached to a Cys, Lys, Orn, homo-Cys, or Ac-Phe,which is attached to the hydrophilic moiety.

Alternatively, the acylated glucagon analog can comprise a spacer,wherein the spacer is both acylated and modified to comprise thehydrophilic moiety. Nonlimiting examples of suitable spacers include aspacer comprising one or more amino acids selected from the groupconsisting of Cys, Lys, Orn, homo-Cys, and Ac-Phe.

In some aspects, the GIP agonist peptide in some embodiments areacylated at the same amino acid position where a hydrophilic moiety islinked, or at a different amino acid position. Nonlimiting examplesinclude acylation at position 9, 10, 12, 13, 14, 16, 17, 20, or 40(e.g., at one or more of positions 10, 14, and 40) and pegylation at oneor more positions in the C-terminal portion of the glucagon analog,e.g., position 24, 28 or 29, within a C-terminal extension (e.g.,37-43), or at the C-terminus (e.g., through adding a C-terminal Cys).

In some specific embodiments, the GIP agonist peptide comprising anacylated amino acid lacks an intramolecular bridge, e.g., a covalentintramolecular bridge (e.g., a lactam bridge).

Amino Acids Comprising a Non-Native Alkyl Group

In accordance with some or any embodiments, the GIP agonist peptideswhich are analogs of glucagon (SEQ ID NO: 1) comprise an amino acidcomprising a non-native alkyl group (referred to herein as an “alkylatedamino acid,” regardless of how it is prepared, e.g., by incorporation ofa previously-alkylated amino acid into the peptide, or alkylation of thepeptide after synthesis). In some or any aspects, the alkylated aminoacid is located at any of positions 9, 10, 12, 13, 14, 16, 17, 20, 37,38, 39, 40, 41, 42, or 43 (e.g., at one or more of positions 10, 14, and40). In exemplary aspects, the alkylated amino acid is located at any ofpositions 9, 10, 12, 16, 20, or 40 or at any of positions 10, 13, 14,16, 17, or 40. In exemplary aspects, the alkylated amino acid is locatedat any of positions 10, 12, 16, or 40 or at any of positions 10, 12, or16. In exemplary aspects, the alkylated amino acid is located at any oneor more of positions 10, 14, and 40.

The alkylated amino acid in some embodiments causes the GIP agonistpeptide to have one or more of (i) a prolonged half-life in circulation,(ii) a delayed onset of action, (iii) an extended duration of action,(iv) an improved resistance to proteases, such as DPP-IV, and (v)increased potency at any one or more of the GIP receptor, GLP-1receptor, and glucagon receptor.

Direct Alkylation

In some embodiments, the alkyl group is directly linked to an amino acidof the GIP agonist peptide. In accordance with one embodiment, the GIPagonist peptide comprises an alkyl group which is attached to thepeptide via an ether, thioether, or amine linkage.

In specific aspects, the GIP agonist peptide comprises an alkyl groupupon direct alkylation of an amine, hydroxyl, or thiol of a side chainof an amino acid of the GIP agonist peptide. In some or any embodiments,the alkyl group is linked to the amino acid of the GIP agonist peptideby reacting the amine, hydroxyl, or thiol with an activated alkyl group.Alkyl groups in some aspects are activated with a leaving group, forexample, a halogen, sulfonate ester, pyridylthiol, ammonium salt, orphenoxyl.

In some or any embodiments, the alkylated amino acid is located at oneof positions 9, 10, 12, 13, 14, 16, 17, 20, or 40 (e.g., at any one ofpositions 10, 14, and 40) or at one of positions 10, 12, or 16. In thisregard, the GIP agonist peptide comprises the amino acid sequence of SEQID NO: 1, or a modified amino acid sequence thereof, comprising one ormore of the amino acid modifications described herein, wherein at leastone of the amino acids at positions 9, 10, 12, 13, 14, 16, 17, 20, and40 (e.g., at any one of positions 10, 14, and 40) is an amino acidcomprising a side chain amine, hydroxyl, or thiol.

In some embodiments, the amino acid comprising a side chain amine is anamino acid of Formula I. In some embodiments, the amino acid of FormulaI, is the amino acid wherein n is 4 (Lys) or n is 3 (Orn). In someembodiments, the amino acid comprising a side chain amine is an aromaticamino acid comprising a side chain amine. In exemplary aspects, thearomatic amino acid comprising a side chain amine is4-amino-phenylalanine (4-aminoPhe), p-amino phenylglycine, p-aminohomophenylalanine, or 3-amino tyrosine. In exemplary aspects, thearomatic amino acid comprising a side chain amine is 4-amino-Phe.

In other embodiments, the amino acid comprising a side chain hydroxyl isan amino acid of Formula II. In some exemplary embodiments, the aminoacid of Formula II is the amino acid wherein n is 1 (Ser). In exemplaryaspects, the amino acid of Formula II is the amino acid wherein n is 2(homoserine). In similar exemplary embodiments, the amino acidcomprising a side chain hydroxyl is a Thr or homothreonine. In similarexemplary embodiments, the amino acid comprising a side chain hydroxylis an aromatic amino acid comprising a side chain hydroxyl. In exemplaryaspects, the aromatic amino acid comprising a side chain hydroxyl istyrosine, homotyrosine, methyl-tyrosine, or 3-amino tyrosine.

In yet other embodiments, the amino acid comprising a side chain thiolis an amino acid of Formula III. In some exemplary embodiments, theamino acid of Formula III is the amino acid wherein n is 1 (Cys). Insome or any exemplary embodiments, the amino acid of Formula III iscovalently attached to an alkyl group, e.g., a non-functionalized orfunctionalized carbon chain. In exemplary aspects, the amino acid isS-palmityl-alkylated (i.e. S-palmitate-alkylated) in which the sulfuratom of a Cys residue is covelantly bound to the δ carbon of palmitate.In other embodiments, the amino acid of Formula III is covalently boundto the (3 carbon of a Cn acetate through the sulfur atom of a Cysresidue, wherein n is an integer from 4 to 30. Examples of differentways to S-palmityl alkylate are shown in FIGS. 6 and 7.

In yet other embodiments, the amino acid comprising a side chain amine,hydroxyl, or thiol is a disubstituted amino acid comprising the samestructure of Formula I, Formula II, or Formula III, except that thehydrogen bonded to the alpha carbon of the amino acid of Formula I,Formula II, or Formula III is replaced with a second side chain.

Alkylation Spacers

In alternative embodiments, the non-native alkyl group is linked via aspacer to an amino acid of the GIP agonist peptide, wherein the spaceris positioned between the amino acid of the GIP agonist peptide and thenon-native alkyl group. In some embodiments, the GIP agonist peptide iscovalently bound to the spacer, which is covalently bound to thenon-native alkyl group.

In exemplary embodiments, the glucagon analog is modified to comprise anon-native alkyl group by alkylation of an amine, hydroxyl, or thiol ofa spacer, which spacer is attached to a side chain of an amino acid atone of positions 9, 10, 12, 13, 14, 16, 17, 20, or 37-43 (e.g., at anyone of positions 10, 14, and 40). In exemplary embodiments, the alkylgroup is attached to the spacer by reacting the amine, hydroxyl, orthiol of the spacer with an alkyl group that has a leaving group (e.g.,halogen, sulfonate ester, pyridylthiol, ammonium salt, phenoxyl).

The amino acid to which the spacer is attached can be any amino acidcomprising a moiety which permits linkage to the spacer. For example, anamino acid comprising a side chain NH₂, —OH, or —COOH (e.g., Lys, Orn,Ser, Asp, or Glu) is suitable. In this respect, the alkylated glucagonanalog can comprise a modified amino acid sequence of SEQ ID NO: 1,comprising one or more of the amino acid modifications described herein,with at least one of the amino acids at positions 9, 10, 12, 13, 14, 16,17, 20, or 37-43 (e.g., at any one or more of positions 10, 14, and 40).modified to any amino acid comprising a side chain amine, hydroxyl, orcarboxylate.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol, or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol. The aminoacid to which the spacer is attached can be any amino acid (e.g., asingly or doubly α-substituted amino acid) comprising a moiety whichpermits linkage to the spacer. For example, an amino acid comprising aside chain NH₂, —OH, or —COOH (e.g., Lys, Orn, Ser, Asp, or Glu) issuitable. In this respect, the GIP agonist peptide in some aspectscomprises the amino acid sequence of SEQ ID NO: 1, or a modified aminoacid sequence thereof comprising one or more of the amino acidmodifications described herein, wherein at least one of the amino acidsat positions 9, 10, 12, 13, 14, 16, 17, 20, and 37-43 (e.g., at any oneof positions 10, 14, and 40) is an amino acid comprising a side chainamine, hydroxyl, or carboxylate.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol, or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol.

When the alkyl group is attached through an amine group of a spacer, thealkyl group in some aspects is attached through the alpha amine orthrough a side chain amine of the spacer amino acid. In the instance inwhich the alkyl group is attached via an alpha amine, the amino acid ofthe spacer can be any amino acid. For example, the amino acid of thespacer can be a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu, Ile,Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5-aminovaleric acid,7-aminoheptanoic acid, and 8-aminooctanoic acid. Alternatively, in someaspects, the amino acid of the spacer is an acidic residue, e.g., Asp,Glu, homoglutamic acid, homocysteic acid, cysteic acid, gamma-glutamicacid.

In the instance in which the alkyl group is attached through a sidechain amine of the amino acid spacer, the spacer is an amino acidcomprising a side chain amine, e.g., an amino acid of Formula I (e.g.,Lys or Orn). In this instance, it is possible for both the alpha amineand the side chain amine of the amino acid of the spacer to be attachedto an alkyl group, such that the GIP agonist peptide is dialkylated.Embodiments of the invention include such dialkylated molecules. In someembodiments, the alkyl group is attached to a 4-amino-Phe, p-aminophenylglycine, p-amino homophenylalanine, or 3-amino tyrosine.

When the alkyl group is attached through a hydroxyl group of a spacer,the amino acid or one of the amino acids of the dipeptide or tripeptidecan be an amino acid of Formula II. In a specific exemplary embodiment,the amino acid is Ser. In similar exemplary embodiments, the alkyl groupis attached to a Thr or homothreonine. In similar exemplary embodiments,the alkyl group is attached via the hydroxyl of an aromatic amino acidcomprising a side chain hydroxyl, e.g., tyrosine, homotyrosine,methyl-tyrosine, or 3-amino tyrosine.

When the alkyl group is attached through a thiol group of a spacer, theamino acid or one of the amino acids of the dipeptide or tripeptide canbe an amino acid of Formula III. In a specific exemplary embodiment, theamino acid is Cys. When the alkyl group is attached through a thiolgroup of a spacer, the amino acid or one of the amino acids of thedipeptide or tripeptide can be an amino acid of Formula III. In aspecific exemplary embodiment, the amino acid is Cys. In exemplaryembodiments, the spacer is a Cys residue, which is covalently attachedto an alkyl group, e.g., a non-functionalized or functionalized carbonchain. In exemplary aspects, the Cys residue is S-palmityl alkylated(i.e., S-palmitate alkylated), optionally, wherein the Cys residue isattached to a Lys residue which is part of the peptide backbone. Inalternative embodiments, the spacer is a dipeptide comprising a Cysresidue, which is covalently attached to an alkyl group. In exemplaryaspects, the Cys is S-palmityl alkylated, and the Cys is attached toanother amino acid of the spacer, which, in turn, is attached to, e.g.,a Lys residue which is part of the peptide backbone. Furtherexemplification of S-palmityl alkylation is provided herein in Example20.

In other exemplary embodiments, the spacer is a bifunctional spacercomprising (i) a first end comprising a leaving group that reacts withan alkyl group that comprises an amine, hydroxyl, or thiol and (ii) asecond end comprising a functional group that reacts with the side chainof the amino acid of the glucagon analog. In exemplary aspects, theamino acid of the glucagon analog is an amino acid of Formula I (e.g.,Lys) or Formula II (e.g., Ser) and the amino acid is functionalized witha carboxylic acid or carboxylic acid derivative. In alternativeexemplary aspects, the amino acid of the glucagon analog is an aminoacid of Formula III and the amino acid is functionalized with ahaloacetabmide, maleimido, or disulfide. In some embodiments, the aminoacid of the glucagon analog is an amino acid comprising a side chaincarboxylate, e.g., Glu, Asp, functionalized with an amine, hydroxyl, orthiol.

In some or any embodiments, the spacer is a hydrophilic bifunctionalspacer. In exemplary embodiments, the hydrophilic bifunctional spacercomprises two or more reactive groups, e.g., an amine, a hydroxyl, athiol, and a carboxyl group or any combinations thereof. In exemplaryembodiments, the hydrophilic bifunctional spacer comprises a hydroxylgroup and a carboxylate. In other embodiments, the hydrophilicbifunctional spacer comprises an amine group and a carboxylate. In otherembodiments, the hydrophilic bifunctional spacer comprises a thiol groupand a carboxylate. In a specific embodiment, the spacer comprises anamino poly(alkyloxy)carboxylate. In this regard, the spacer cancomprise, for example, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is anyinteger from 1 to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.).

In exemplary embodiments, the spacer comprises a small polyethyleneglycol moiety (PEG) comprising a structure [—O—CH₂—CH₂—]_(n), wherein nis an integer between 2 and 16, (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16). Such small PEGs are referred to herein as a“miniPEG.” In exemplary aspects, the miniPEG is a functionalized miniPEGcomprising one or more functional groups. Suitable functional groupsinclude, but are not limited to, an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In exemplary aspects, theminiPEG is a miniPEG acid comprising a structure{[—O—CH₂—CH₂—]_(n)—COO—}, wherein n is defined as above. In exemplaryaspects, the miniPEG is an amido miniPEG comprising a structure{—N—CH₂—CH₂—[—O—CH₂—CH₂—]_(n)}, wherein n is defined as above. Inexemplary aspects, the miniPEG is an amido miniPEG acid comprising astructure {—N—CH₂—CH₂—[—O—CH₂—CH₂—]_(n)—COO—}, wherein n is defined asabove. Suitable reagents for use in alkylating an amino acid with aminiPEG are commercially available from vendors, such as PeptidesInternational (Louisville, Ky.).

In some embodiments, the spacer is a hydrophobic bifunctional spacer.Hydrophobic bifunctional spacers are known in the art. See, e.g.,Bioconjugate Techniques, G. T. Hermanson (Academic Press, San Diego,Calif., 1996), which is incorporated by reference in its entirety. Inexemplary embodiments, the hydrophobic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In exemplary embodiments,the hydrophobic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydrophobic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydrophobic bifunctional spacer comprises a thiol group and acarboxylate. Suitable hydrophobic bifunctional spacers comprising acarboxylate and a hydroxyl group or a thiol group are known in the artand include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoicacid.

In some embodiments, the bifunctional spacer is not a dicarboxylic acidcomprising an unbranched, methylene of 1-7 carbon atoms between thecarboxylate groups. In some embodiments, the bifunctional spacer is adicarboxylic acid comprising an unbranched, methylene of 1-7 carbonatoms between the carboxylate groups.

The spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilicbifunctional spacer, or hydrophobic bifunctional spacer) in specificembodiments is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or10 atoms) in length. In more specific embodiments, the spacer is about 3to 10 atoms (e.g., 6 to 10 atoms) in length and the non-native alkylgroup is a C12 to C18 alkyl, e.g., C14 alkyl, C16 alkyl, such that thetotal length of the spacer and alkyl group is 14 to 28 atoms, e.g.,about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28atoms. In some embodiments, the length of the spacer and alkyl group is17 to 28 (e.g., 19 to 26, 19 to 21) atoms.

In accordance with some or any of the foregoing embodiments, thebifunctional spacer can be a synthetic or naturally occurring amino acid(including, but not limited to, any of those described herein)comprising an amino acid backbone that is 3 to 10 atoms in length (e.g.,6-amino hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoic acid, and8-aminooctanoic acid). Alternatively, the spacer can be a dipeptide ortripeptide spacer having a peptide backbone that is 3 to 10 atoms (e.g.,6 to 10 atoms) in length. Each amino acid of the dipeptide or tripeptidespacer can be the same as or different from the other amino acid(s) ofthe dipeptide or tripeptide and can be independently selected from thegroup consisting of: naturally-occurring or coded and/or non-coded ornon-naturally occurring amino acids, including, for example, any of theD or L isomers of the naturally-occurring amino acids (Ala, Cys, Asp,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val,Trp, Tyr), or any D or L isomers of the non-naturally occurring ornon-coded amino acids selected from the group consisting of: β-alanine(β-Ala), N-α-methyl-alanine (Me-Ala), aminobutyric acid (Abu),γ-aminobutyric acid (γ-Abu), aminohexanoic acid (ε-Ahx), aminoisobutyricacid (Aib), aminomethylpyrrole carboxylic acid,aminopiperidinecarboxylic acid, aminoserine (Ams),aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methylamide, β-aspartic acid (β-Asp), azetidine carboxylic acid,3-(2-benzothiazolyl)alanine, α-tert-butylglycine,2-amino-5-ureido-n-valeric acid (citrulline, Cit), β-Cyclohexylalanine(Cha), acetamidomethyl-cysteine, diaminobutanoic acid (Dab),diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA),dimethylthiazolidine (DMTA), γ-Glutamic acid (γ-Glu), homoserine (Hse),hydroxyproline (Hyp), isoleucine N-methoxy-N-methyl amide,methyl-isoleucine (MeIle), isonipecotic acid (Isn), methyl-leucine(MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine,methanoproline, methionine-sulfoxide (Met(O)), methionine-sulfone(Met(O)₂), norleucine (Nle), methyl-norleucine (Me-Nle), norvaline(Nva), ornithine (Orn), para-aminobenzoic acid (PABA), penicillamine(Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4-Cl)),4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-NO₂)),4-cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg),piperidinylalanine, piperidinylglycine, 3,4-dehydroproline,pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec),O-Benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta),4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA),4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA),1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid (Tic),tetrahydropyranglycine, thienylalanine (Thi), O-benzyl-phosphotyrosine,O-Phosphotyrosine, methoxytyrosine, ethoxytyrosine,O-(bis-dimethylamino-phosphono)-tyrosine, tyrosine sulfatetetrabutylamine, methyl-valine (MeVal), and alkylated3-mercaptopropionic acid. In exemplary aspects, the spacer is a Cysresidue.

In some embodiments, the spacer comprises an overall negative charge,e.g., comprises one or two negative-charged amino acids. In someembodiments, the dipeptide is not any of the dipeptides of generalstructure A-B, wherein A is selected from the group consisting of Gly,Gln, Ala, Arg, Asp, Asn, Ile, Leu, Val, Phe, and Pro, wherein B isselected from the group consisting of Lys, His, Trp. In someembodiments, the amino acids of the dipeptide spacer are selected fromthe group consisting of: Ala, β-Ala, Leu, Pro, γ-aminobutyric acid, Glu,and γ-Glu.

In some exemplary embodiments, the GIP agonist peptide comprises analkyl group upon alkylation of an amine, hydroxyl, or thiol of a spacer,which spacer is attached to a side chain of an amino acid at position 9,10, 12, 13, 14, 16, 17, 20, or 37-43, (e.g., at any one or more ofpositions 10, 14, and 40), or at the C-terminal amino acid of the GIPagonist peptide.

In yet more specific embodiments, the alkyl group is attached to theamino acid at any of positions 9, 10, 12, 13, 14, 16, 17, 20, or 37-43(e.g., at any one or more of positions 10, 14, and 40), of the peptideanalog and the length of the spacer and alkyl group is 14 to 28 atoms.The amino acid at any of positions 9, 10, 12, 13, 14, 16, 17, 20, or37-43, (e.g., at any one or more of positions 10, 14, and 40), in someaspects, is an amino acid of Formula I, e.g., Lys, or a disubstitutedamino acid related to Formula I. In more specific embodiments, thepeptide analog lacks an intramolecular bridge, e.g., a covalentintramolecular bridge. The glucagon analog, for example, can be aglucagon analog comprising one or more alpha, alpha-disubstituted aminoacids, e.g., AIB, for stabilizing the alpha helix of the analog.

Alkyl Groups

The non-native alkyl group of the alkylated amino acid can be of anysize, e.g., any length carbon chain, and can be linear or branched. Insome specific embodiments, the alkyl group is a C4 to C30 alkyl. Forexample, the alkyl group can be any of a C4 alkyl, C6 alkyl, C8 alkyl,C10 alkyl, C12 alkyl, C14 alkyl, C16 alkyl, C18 alkyl, C20 alkyl, C22alkyl, C24 alkyl, C26 alkyl, C28 alkyl, or a C30 alkyl. In someembodiments, the alkyl group is a C8 to C20 alkyl, e.g., a C12 to C18,alkyl, a C14 alkyl or a C16 alkyl. In exemplary embodiments, the alkylgroup is a C16 to C30 alkyl, e.g., a C20 to C28 alkyl, a C16 to C22alkyl.

In exemplary embodiments, the non-native alkyl group of the alkylatedamino acid is a functionalized linear or branched carbon chain of anylength. In some specific embodiments, the carbon chain is a C4 to C30alkyl. For example, the alkyl group can be any of a C4 alkyl, C6 alkyl,C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, C16 alkyl, C18 alkyl, C20alkyl, C22 alkyl, C24 alkyl, C26 alkyl, C28 alkyl, or a C30 alkyl. Insome embodiments, the alkyl group is a C8 to C20 alkyl, e.g., a C12 toC18, alkyl, a C14 alkyl or a C16 alkyl. In exemplary embodiments, thealkyl group is a C16 to C30 alkyl, e.g., a C20 to C28 alkyl, a C16 toC22 alkyl. In exemplary aspects, the functionalized carbon chaincomprises a functional group, including, but not limited, carboxy,sulfhydryl, amine, ketyl, sulfoxyl or amido.

In exemplary embodiments, the non-native alkyl group is acarboxy-functionalized carbon chain of structure -Cx-COOH, wherein x isan integer, optionally an integer between 4-30 (e.g., 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30), wherein the carboxy carbon is the alpha carbon and each ofthe carbons of Cx are designated beta, gamma, delta, epsilon, etc.,wherein the beta carbon is attached to the alpha carbon. For example,wherein, when x is 4, the non-native alkyl group would be designated asfollows: C_(ε)—C_(δ)—C_(γ)—C_(β)—C_(α)OOH. In exemplary embodiments, thecarboxy-functionalized carbon chain is attached via a carbon other thanthe carboxy carbon, i.e., one of the carbons of Cx. In exemplaryaspects, the carboxy-functionalized carbon chain is attached via thebeta, gamma, delta, or epsilon carbon of the carboxy-functionalizedcarbon chain to the side chain of the alkylated amino acid. Inalternative embodiments, the carboxy-functionalized carbon chain isattached via the beta, gamma, delta, or epsilon carbon of thecarboxy-functionalized carbon chain to the side chain of a spacer whichspacer is attached to the alkylated amino acid. In exemplary aspects,the carboxy-functionalized carbon chain is attached via the beta carbonof the carboxy-functionalized carbon chain to the side chain of thealkylated amino acid. In alternative embodiments, thecarboxy-functionalized carbon chain is attached via the beta carbon ofthe carboxy-functionalized carbon chain to the side chain of a spacerwhich spacer is attached to the alkylated amino acid.

Methods of Attaching an Alkyl Group

Methods of attaching an alkyl group to an amino acid are known in theart. For example, an alkyl groups activated with a leaving group may bereacted with an amino acid comprising a nucleophilic side chain, e.g., aside chain comprising an amine, hydroxyl, or thiol. The leaving group inexemplary aspects is a halogen, sulfonate ester, pyridylthiol, ammoniumsalt, or phenoxyl.

In exemplary embodiments, the amino acid to be attached to an alkylgroup is a Cys residue and the sulfur atom is alkylated, e.g.,“S-alkylated.” In exemplary embodiments, the sulfur of the Cys isreacted with the leaving group of an alkyl group comprising acarboxy-functionalized carbon chain of structure -Cx-COOH, wherein x isan integer, optionally an integer between 4-30 (e.g., 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30), wherein the carboxy carbon is the alpha carbon and each ofthe carbons of Cx are designated beta, gamma, delta, epsilon, etc.,wherein the beta carbon is attached to the alpha carbon. For example,wherein, when x is 4, the non-native alkyl group would be designated asfollows: C_(ε)—C_(δ)—C_(γ)—C_(β)—C_(α)OOH. In exemplary embodiments, thecarboxy-functionalized carbon chain is attached via a carbon other thanthe carboxy carbon, i.e., one of the carbons of Cx. In exemplaryaspects, the carboxy-functionalized carbon chain is attached via thebeta, gamma, delta, or epsilon carbon of the carboxy-functionalizedcarbon chain to the side chain of the alkylated amino acid. Inalternative embodiments, the carboxy-functionalized carbon chain isattached via the beta, gamma, delta, or epsilon carbon of thecarboxy-functionalized carbon chain to the side chain of a spacer whichspacer is attached to the alkylated amino acid. In exemplary aspects,the carboxy-functionalized carbon chain is attached via the beta carbonof the carboxy-functionalized carbon chain to the side chain of thealkylated amino acid. In alternative embodiments, thecarboxy-functionalized carbon chain is attached via the beta carbon ofthe carboxy-functionalized carbon chain to the side chain of a spacerwhich spacer is attached to the alkylated amino acid.

In exemplary aspects, the leaving group is a halogen, such as iodine,bromine, chlorine, or fluorine, sulfonate esters such as tosylate,triflates, or fluorosulfonates, pyridylthiol, ammonium salt, diazoniumsalts, nitrates, phosphates or phenoxyl.

In specific aspects, the alkyl group comprises an iodine leaving groupand a carboxy-functionalized carbon chain comprising a total of 16carbons (including the carbon of the carboxylate). Alkylation with suchan iodo-carboxylic acid may be referred to as “S-palmityl alkylation”which is synonymous with “S-palmitate alkylation.” Furtherexemplification of S-palmityl alkylation is provided herein in Examples1 and 20.

Additional Alkyl Groups

The peptide in some aspects comprises an alkylated amino acid at aposition other than positions 9, 10, 12, 13, 14, 16, 17, 20, or 37-43(e.g., at any one or more of positions 10, 14, and 40). The location ofthe alkylated amino acid may be any position within the GIP agonistpeptide, including any of positions 1-29, a position C-terminal to the29^(th) amino acid (e.g., the amino acid at position 30, 31, 32, 33, 34,35, 36, 44, 45, 46, 47, etc., at a position within a C-terminalextension or at the C-terminus), optionally together with a secondalkylated amino acid at any of positions 9, 10, 12, 13, 14, 16, 17, 20,or 37-43 (e.g., at any one or more of positions 10, 14, and 40),provided that the GIP agonist activity of the peptide analog isretained, if not enhanced. Nonlimiting examples include positions 5, 7,11, 13, 14, 17, 18, 19, 21, 24, 27, 28, or 29.

Consistent with the foregoing, the glucagon analog, in exemplaryaspects, comprises two (or more) alkylated amino acids. In exemplaryaspects, all of the alkylated amino acids are located at two positionsof positions 9, 10, 12, 13, 14, 16, 17, 20, or 37-43 (e.g., at any oneor more of positions 10, 14, and 40). In exemplary aspects, the peptidecomprising a first alkylated amino acid at position 10 and a secondalkylated amino acid at position 40.

In yet additional exemplary embodiments, the glucagon analog comprisesadditional alkyl groups attached to one amino acid of the peptidebackbone. The two (or more) alkyl groups may be the same alkyl group ordifferent alkyl groups, arranged in a branched or linear formation. Forexample, to achieve a branched formation, the glucagon analog maycomprise one alkylated amino acid (which is part of the peptidebackbone) attached to a spacer comprising at least three functionalgroups—at least two of which are each covalently attached to an alkylgroup and one of which is attached to the alkylated amino acid of thepeptide backbone. In exemplary aspects, a branched formation may beachieved through, e.g., a Lys residue comprising two amine groups (aside chain amine and an alpha amine) for direct attachment to a fattyalkyl group or indirect attachment to a fatty alkyl group via a spacer.In exemplary aspects, an additional spacer may be placed between theamino acid of the peptide backbone and the spacer comprising at leastthree functional groups. For example, the amino acid of the peptidebackbone may be attached (e.g., via its side chain) to a first spacer,which, in turn, is attached to a second spacer, wherein the secondspacer comprises at least three functional groups—at least two of whichare each covalently attached to an alkyl group and one of which isattached to the first spacer.

In exemplary aspects, wherein the alkyl groups are arranged in a linearformation, the glucagon analog comprises one alkylated amino acid (whichis part of the peptide backbone) directly attached to a first alkylgroup, which, in turn, is attached to a spacer, which, in turn, isattached to a second alkyl group.

Exemplary structures of dual alkylated compounds are derivable from thedual acylated compounds depicted in FIG. 5A.

Hydrophilic Moieties and Alkyl Groups

The GIP agonist peptides comprising an alkylated amino acid optionallyfurther comprises a hydrophilic moiety. In some specific embodiments thehydrophilic moiety comprises a polyethylene glycol (PEG) chain. Theincorporation of a hydrophilic moiety in some aspects is accomplishedthrough any suitable means, such as any of the methods described herein.In this regard, the GIP agonist peptide can comprise SEQ ID NO: 1,including any of the modifications described herein, in which at leastone of the amino acids at any of positions 9, 10, 12, 13, 14, 16, 17,20, and 37-43 (e.g., at any one or more of positions 10, 14, and 40) ofthe GIP agonist peptide comprises an alkyl group and at least one of theamino acids at position 16, 17, 21, 24, or 29, a position within aC-terminal extension, or the C-terminal amino acid is a Cys, Lys, Orn,homo-Cys, or Ac-Phe, of which the side chain is covalently bonded to ahydrophilic moiety (e.g., PEG). In some embodiments, the amino acid atany of positions 9, 10, 12, 13, 14, 16, 17, 20, and 37-43 (e.g., at anyone or more of positions 10, 14, and 40) of the GIP agonist peptide isattached (optionally via a spacer) to the alkyl group and that aminoacid is further attached to a hydrophilic moiety or is further attachedto a Cys, Lys, Orn, homo-Cys, or Ac-Phe, which is attached to thehydrophilic moiety.

Alternatively, the alkylated glucagon analog can comprise a spacer,wherein the spacer is both alkylated and modified to comprise thehydrophilic moiety. Nonlimiting examples of suitable spacers include aspacer comprising one or more amino acids selected from the groupconsisting of Cys, Lys, Orn, homo-Cys, and Ac-Phe.

In some aspects, the GIP agonist peptide in some embodiments arealkylated at the same amino acid position where a hydrophilic moiety islinked, or at a different amino acid position. Nonlimiting examplesinclude alkylation at position 9, 10, 12, 13, 14, 16, 17, 20, or 40(e.g., at any one or more of positions 10, 14, and 40) and pegylation atone or more positions in the C-terminal portion of the glucagon analog,e.g., position 24, 28 or 29, within a C-terminal extension (e.g.,37-43), or at the C-terminus (e.g., through adding a C-terminal Cys).

Additional Alkyl Group Embodiment

In specific embodiments, the GIP agonist peptide comprising an alkylatedamino acid lacks an intramolecular bridge, e.g., a covalentintramolecular bridge (e.g., a lactam bridge).

Stabilization of the Alpha Helix and Alpha Helix Stabilizing Amino Acids

Without being bound to any particular theory, the GIP agonist peptideswhich are glucagon analogs comprise a helical structure, e.g., an alphahelix. In some or any embodiments, the GIP agonist peptide comprisesamino acids which stabilize the alpha helical structure. Accordingly, insome aspects, the GIP agonist peptide comprises one or more alpha helixstabilizing amino acids. As used herein, the term “alpha helix promotingamino acid” is used interchangeably with the term “alpha helixstabilizing amino acid” and refers to an amino acid which providesincreased stability to an alpha helix of the GIP agonist peptides ofwhich it is a part. Alpha helix promoting amino acids are known in theart. See, for example, Lyu et al., Proc Natl Acad Sci U.S.A. 88:5317-5320 (1991); Branden & Tooze, Introduction to Protein Structure,Garland Publishing, New York, N.Y., 1991; Fasman, Prediction of ProteinStructure and the Principles of Protein Conformation, ed. Fasman,Plenum, N.Y., 1989). Suitable alpha helix promoting amino acids forpurposes herein include, but are not limited to: alanine, norvaline,norleucine, alpha aminobutyric acid, alpha-aminoisobutyric acid (AIB),leucine, isoleucine, valine, and the like. In some embodiments, thealpha helix promoting amino acid is any amino acid which is part of analpha helix found in a naturally-occurring protein, e.g., Leu, Phe, Ala,Met, Gly, Ile, Ser, Asn, Glu, Asp, Lys, Arg.

In exemplary embodiments, the alpha helix promoting amino acid providesmore stability to the alpha helix as compared to glycine or alanine. Inexemplary embodiments, the alpha helix promoting amino acid is an alpha,alpha di-substituted amino acid, e.g., AIB.

Alpha Helix: Position of Alpha Helix Promoting Amino Acids

In some or any embodiments of the present disclosures, the GIP agonistpeptide comprises an amino acid sequence which is similar to nativeglucagon (SEQ ID NO: 1) and the GIP agonist peptide comprises at leastone alpha helix promoting amino acid at one or more of positions 16-21of the peptide analog (e.g., one or more of positions 16, 17, 18, 19,20, 21). In some or any embodiments, the peptide analog comprises analpha helix promoting amino acid at one, two, three, or all of positions16, 17, 20, and 21.

Alpha Helix: Alpha, Alpha Di-Substituted Amino Acids

In some or any embodiments, the alpha helix promoting amino acid is analpha,alpha di-substituted amino acid. In specific embodiments, thealpha, alpha di-substituted amino acid comprises R¹ and R², each ofwhich is bonded to the alpha carbon, wherein each of R¹ and R² isindependently selected from the group consisting of C1-C4 alkyl,optionally substituted with a hydroxyl, amide, thiol, halo, or R¹ and R²together with the alpha carbon to which they are attached form a ring(e.g., a C3-C8 ring). In exemplary embodiments, each of R¹ and R² isselected from the group consisting of: methyl, ethyl, propyl, andn-butyl, or R¹ and R² together form a cyclooctane or cycloheptane (e.g.,1-aminocyclooctane-1-carboxylic acid). In exemplary embodiments, R¹ andR² are the same. In other embodiments, R¹ is different from R². Inexemplary aspects, each of R¹ and R² is a C1-C4 alkyl. In some aspects,each of R¹ and R² is a C1 or C2 alkyl. In exemplary embodiments, each ofR¹ and R² is methyl, such that the alpha, alpha disubstituted amino acidis alpha-aminoisobutyric acid (AIB). In other exemplary embodiments, thealpha, alpha disubstituted amino acid is ACPC.

In some aspects, the GIP agonist peptide described herein comprises oneor more alpha, alpha di-substituted amino acids and the GIP agonistpeptide specifically lacks a covalent intramolecular bridge (e.g., alactam), since the alpha, alpha disubstituted amino acid is capable ofstabilizing the alpha helix in the absence of a covalent bridge. In someaspects, the GIP agonist peptide comprises one or more alpha, alphadi-substituted amino acids at the C-terminus (around positions 12-29).In some or any embodiments, one, two, three, four or more of positions16, 17, 18, 19, 20, 21, 24 or 29 or, one, two, three, four, five, or allof positions 16, 17, 18, 19, 20, or 21 of the GIP agonist peptide issubstituted with an α,α-disubstituted amino acid, e.g., aminoiso-butyric acid (AIB), an amino acid disubstituted with the same or adifferent group selected from methyl, ethyl, propyl, and n-butyl, orwith a cyclooctane or cycloheptane (e.g.,1-aminocyclooctane-1-carboxylic acid). For example, substitution ofposition 20 with AIB enhances GIP activity, in the absence of anintramolecular bridge, e.g., a non-covalent intramolecular bridge (e.g.,a salt bridge) or a covalent intramolecular bridge (e.g., a lactam). Insome or any embodiments, one, two, three or more of positions 16, 20, 21or 24 are substituted with AIB. In exemplary embodiments, one, two orall of the amino acids corresponding to positions 2, 16, and 20 ofnative human glucagon (SEQ ID NO: 1) are substituted with an alpha,alpha disubstituted amino acid such as AIB. In exemplary aspects, theglucagon analog comprises an AIB at positions 2 and 16 or at positions 2and 20. In other exemplary aspects, the glucagon analog comprises aD-Ser at position 2 and an AIB at position 16 or position 20.

Alpha Helix: Intramolecular Bridges

In some exemplary embodiments, the alpha helix promoting amino acid isan amino acid which is linked to another amino acid of the GIP agonistpeptide via an intramolecular bridge. In such embodiments, each of thesetwo amino acids linked via an intramolecular bridge is considered analpha helix promoting amino acid. In exemplary embodiments, the GIPagonist peptide comprises one or two intramolecular bridges. Inexemplary embodiments, the GIP agonist peptide comprises oneintramolecular bridge in combination with at least one other alpha helixpromoting amino acid, e.g., an alpha, alpha-disubstituted amino acid.

In some embodiments, the intramolecular bridge is a bridge whichconnects two parts of the GIP agonist peptide via noncovalent bonds,including, for example, van der Waals interactions, hydrogen bonds,ionic bonds, hydrophobic interactions, dipole-dipole interactions, andthe like. In this regard, the glucagon analog comprises a non-covalentintramolecular bridge. In some embodiments, the non-covalentintramolecular bridge is a salt bridge.

In some embodiments, the intramolecular bridge is a bridge whichconnects two parts of the GIP agonist peptide via covalent bonds. Inthis regard, the GIP agonist peptide comprises a covalent intramolecularbridge.

In some embodiments, the intramolecular bridge (e.g., non-covalentintramolecular bridge, covalent intramolecular bridge) is formed betweentwo amino acids that are 3 amino acids apart, e.g., amino acids atpositions i and i+4, wherein i is any integer between 12 and 25 (e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25). Moreparticularly, the side chains of the amino acid pairs 12 and 16, 16 and20, 20 and 24 or 24 and 28 (amino acid pairs in which i=12, 16, 20, or24) are linked to one another and thus stabilize the alpha helix.Alternatively, i can be 17. In some specific embodiments, the GIPagonist peptide comprises an intramolecular bridge between amino acids17 and 21. In some specific embodiments, the GIP agonist peptidecomprises an intramolecular bridge between the amino acids at positions16 and 20 or 12 and 16 and a second intramolecular bridge between theamino acids at positions 17 and 21. GIP agonist peptides comprising oneor more intramolecular bridges are contemplated herein. In specificembodiments, wherein the amino acids at positions i and i+4 are joinedby an intramolecular bridge, the size of the linker is about 8 atoms, orabout 7-9 atoms.

In other embodiments, the intramolecular bridge is formed between twoamino acids that are two amino acids apart, e.g., amino acids atpositions j and j+3, wherein j is any integer between 12 and 26 (e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26). In somespecific embodiments, j is 17. In specific embodiments, wherein aminoacids at positions j and j+3 are joined by an intramolecular bridge, thesize of the linker is about 6 atoms, or about 5 to 7 atoms.

In yet other embodiments, the intramolecular bridge is formed betweentwo amino acids that are 6 amino acids apart, e.g., amino acids atpositions k and k+7, wherein k is any integer between 12 and 22 (e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22). In some specificembodiments, k is 12, 13, or 17. In an exemplary embodiment, k is 17.

Alpha Helix: Amino Acids Involved in Intramolecular Bridges

Examples of amino acid pairings that are capable of bonding (covalentlyor non-covalently) to form a six-atom linking bridge include Orn andAsp, Glu and an amino acid of Formula I, wherein n is 2, andhomoglutamic acid and an amino acid of Formula I, wherein n is 1,wherein Formula I is:

Examples of amino acid pairings that are capable of bonding to form aseven-atom linking bridge include Orn-Glu (lactam ring); Lys-Asp(lactam); or Homoser-Homoglu (lactone). Examples of amino acid pairingsthat may form an eight-atom linker include Lys-Glu (lactam); Homolys-Asp(lactam); Orn-Homoglu (lactam); 4-aminoPhe-Asp (lactam); or Tyr-Asp(lactone). Examples of amino acid pairings that may form a nine-atomlinker include Homolys-Glu (lactam); Lys-Homoglu (lactam);4-aminoPhe-Glu (lactam); or Tyr-Glu (lactone). Any of the side chains onthese amino acids may additionally be substituted with additionalchemical groups, so long as the three-dimensional structure of thealpha-helix is not disrupted. One of ordinary skill in the art canenvision alternative pairings or alternative amino acid analogs,including chemically modified derivatives, that would create astabilizing structure of similar size and desired effect. For example, ahomocysteine-homocysteine disulfide bridge is 6 atoms in length and maybe further modified to provide the desired effect.

Even without covalent linkage, the amino acid pairings described above(or similar pairings that one of ordinary skill in the art can envision)may also provide added stability to the alpha-helix through non-covalentbonds, for example, through formation of salt bridges orhydrogen-bonding interactions. Accordingly, salt bridges may be formedbetween: Orn and Glu; Lys and Asp; Homo-serine and Homo-glutamate; Lysand Glu; Asp and Arg; Homo-Lys and Asp; Orn and Homo-Glutamate;4-aminoPhe and Asp; Tyr and Asp; Homo-Lys and Glu; Lys and Homo-Glu;4-aminoPhe and Glu; or Tyr and Glu. In some embodiments, the analogcomprises a salt bridge between any of the following pairs of aminoacids: Orn and Glu; Lys and Asp; Lys and Glu; Asp and Arg; Homo-Lys andAsp; Orn and Homo-Glutamate; Homo-Lys and Glu; and Lys and Homo-Glu.Salt bridges may be formed between other pairs of oppositely chargedside chains. See, e.g., Kallenbach et al., Role of the Peptide Bond inProtein Structure and Folding, in The Amide Linkage: StructuralSignificance in Chemistry, Biochemistry, and Materials Science, JohnWiley & Sons, Inc. (2000).

In some embodiments, the non-covalent intramolecular bridge is ahydrophobic bridge. In accordance with one embodiment, the alpha helixof the analog is stabilized through the incorporation of hydrophobicamino acids at positions j and j+3 or i and i+4. For instance, i can beTyr and i+4 can be either Val or Leu; i can be Phe and i+4 can be Met;or i can be Phe and i+4 can be Ile. It should be understood that, forpurposes herein, the above amino acid pairings can be reversed, suchthat the indicated amino acid at position i could alternatively belocated at i+4, while the i+4 amino acid can be located at the iposition. It should also be understood that suitable amino acid pairingscan be formed for j and j+3.

Alpha Helix: Covalent Intramolecular Bridge

In some embodiments, the covalent intramolecular bridge is a lactam ringor lactam bridge. The size of the lactam ring can vary depending on thelength of the amino acid side chains, and in one embodiment the lactamis formed by linking the side chains of an ornithine to a aspartic acidside chain. Lactam bridges and methods of making the same are known inthe art. See, for example, Houston, Jr., et al., J Peptide Sci 1:274-282 (2004), and Example 1 herein. In some embodiments, the analogcomprises a modified sequence of SEQ ID NO: 1 and a lactam bridgebetween i and i+4, wherein i is as defined herein above. In someembodiments, the GIP agonist peptide comprises two lactam bridges: onebetween the amino acids at positions 16 and 20 and another between theamino acids at positions 17 and 21. In some embodiments, the GIP agonistpeptide comprises one lactam bridge and one salt bridge. Furtherexemplary embodiments, are described herein in the section entitled“EXAMPLES.” Further exemplary embodiments include the followingpairings, optionally with a lactam bridge: Glu at position 12 with Lysat position 16; native Lys at position 12 with Glu at position 16; Gluat position 16 with Lys at position 20; Lys at position 16 with Glu atposition 20; Glu at position 20 with Lys at position 24; Lys at position20 with Glu at position 24; Glu at position 24 with Lys at position 28;Lys at position 24 with Glu at position 28.

In some embodiments, the covalent intramolecular bridge is a lactone.Suitable methods of making a lactone bridge are known in the art. See,for example, Sheehan et al., J Am Chem Soc 95: 875-879 (1973).

In some aspects, olefin metathesis is used to cross-link one or twoturns of the alpha helix of the analog using an all-hydrocarboncross-linking system. The GIP agonist peptide in this instance comprisesα-methylated amino acids bearing olefinic side chains of varying lengthand configured with either R or S stereochemistry at the j and j+3 or iand i+4 positions. In some embodiments, the olefinic side comprises(CH₂)n, wherein n is any integer between 1 to 6. In some embodiments, nis 3 for a cross-link length of 8 atoms. In some embodiments, n is 2 fora cross-link length of 6 atoms. Suitable methods of forming suchintramolecular bridges are described in the art. See, for example,Schafineister et al., J. Am. Chem. Soc. 122: 5891-5892 (2000) andWalensky et al., Science 305: 1466-1470 (2004). In alternativeembodiments, the analog comprises O-allyl Ser residues located onadjacent helical turns, which are bridged together viaruthenium-catalyzed ring closing metathesis. Such procedures ofcross-linking are described in, for example, Blackwell et al., Angew,Chem., Int. Ed. 37: 3281-3284 (1998).

In specific aspects, use of the unnatural thio-dialanine amino acid,lanthionine, which has been widely adopted as a peptidomimetic ofcystine, is used to cross-link one turn of the alpha helix. Suitablemethods of lanthionine-based cyclization are known in the art. See, forinstance, Matteucci et al., Tetrahedron Letters 45: 1399-1401 (2004);Mayer et al., J. Peptide Res. 51: 432-436 (1998); Polinsky et al., J.Med. Chem. 35: 4185-4194 (1992); Osapay et al., J. Med. Chem. 40:2241-2251 (1997); Fukase et al., Bull. Chem. Soc. Jpn. 65: 2227-2240(1992); Harpp et al., J. Org. Chem. 36: 73-80 (1971); Goodman and Shao,Pure Appl. Chem. 68: 1303-1308 (1996); and Osapay and Goodman, J. Chem.Soc. Chem. Commun. 1599-1600 (1993).

In some embodiments, α,ω-diaminoalkane tethers, e.g., 1,4-diaminopropaneand 1,5-diaminopentane) between two Glu residues at positions i and i+7are used to stabilize the alpha helix of the analog. Such tethers leadto the formation of a bridge 9-atoms or more in length, depending on thelength of the diaminoalkane tether. Suitable methods of producingpeptides cross-linked with such tethers are described in the art. See,for example, Phelan et al., J. Am. Chem. Soc. 119: 455-460 (1997).

In yet other embodiments, a disulfide bridge is used to cross-link oneor two turns of the alpha helix of the analog. Alternatively, a modifieddisulfide bridge in which one or both sulfur atoms are replaced by amethylene group resulting in an isosteric macrocyclization is used tostabilize the alpha helix of the analog. Suitable methods of modifyingpeptides with disulfide bridges or sulfur-based cyclization aredescribed in, for example, Jackson et al., J. Am. Chem. Soc. 113:9391-9392 (1991) and Rudinger and Jost, Experientia 20: 570-571 (1964).

In yet other embodiments, the alpha helix of the analog is stabilizedvia the binding of metal atom by two His residues or a His and Cys pairpositioned at j and j+3, or i and i+4. The metal atom can be, forexample, Ru(III), Cu(II), Zn(II), or Cd(II). Such methods of metalbinding-based alpha helix stabilization are known in the art. See, forexample, Andrews and Tabor, Tetrahedron 55: 11711-11743 (1999); Ghadiriet al., J. Am. Chem. Soc. 112: 1630-1632 (1990); and Ghadiri et al., J.Am. Chem. Soc. 119: 9063-9064 (1997).

The alpha helix of the GIP agonist peptide can alternatively bestabilized through other means of peptide cyclizing, which means arereviewed in Davies, J. Peptide. Sci. 9: 471-501 (2003). The alpha helixcan be stabilized via the formation of an amide bridge, thioetherbridge, thioester bridge, urea bridge, carbamate bridge, sulfonamidebridge, and the like. For example, a thioester bridge can be formedbetween the C-terminus and the side chain of a Cys residue.Alternatively, a thioester can be formed via side chains of amino acidshaving a thiol (Cys) and a carboxylic acid (e.g., Asp, Glu). In anothermethod, a cross-linking agent, such as a dicarboxylic acid, e.g.,suberic acid (octanedioic acid), etc. can introduce a link between twofunctional groups of an amino acid side chain, such as a free amino,hydroxyl, thiol group, and combinations thereof.

Additional Descriptions

Provided below are additional descriptions of the glucagon analogs ofthe present disclosures. As discussed herein, the position of the aminoacid in the descriptions below is referenced with regard to the aminoacid numbering of SEQ ID NO: 1. Also, while the descriptions below arediscussed in reference to native human glucagon (SEQ ID NO: 1), e.g.,modifications of SEQ ID NO: 1, these descriptions do not necessarilyimply (i) that such modifications are present in all of the presentlydisclosed peptides and (ii) that the only method of making the presentlydisclosed peptides is to start with native human glucagon and modifythat sequence. Rather, the descriptions are provided to describe someembodiments of the glucagon analogs of the present disclosures, and thepeptides of the present disclosures may be made de novo withoututilizing native human glucagon as a starting material, as furtherdescribed in the section entitled “METHODS OF MAKING PEPTIDES.”

Position 1

In some embodiments, the glucagon analog comprises an amino acidmodification at position 1, relative to SEQ ID NO: 1, e.g., the glucagonanalog comprises an amino acid which is not His at position 1. Inexemplary aspects, the glucagon analog comprises a large, aromatic aminoacid at position 1. In exemplary embodiments, the large, aromatic aminoacid is Tyr, Phe, Trp, amino-Phe (e.g., 4-amino-Phe), chloro-Phe,sulfo-Phe, 4-pyridyl-Ala, methyl-Tyr, or 3-amino Tyr.

In other exemplary embodiments, the glucagon analog comprises an aminoacid comprising an imidazole side chain at position 1. In exemplaryaspects, the amino acid at position 1 comprises a structure of FormulaA:

wherein each of R1 and R2 independently is selected from the groupconsisting of H, (C1-6)alkyl, O(C16)alkyl, (C16)alkyl-OH, F, and(C1-C6)alkyl of which at least one H is replaced by F.

In exemplary aspects, the amino acid at position 1 is the native residueof glucagon (SEQ ID NO: 1) L-histidine (His), or is a derivative of His(His derivative), e.g., a derivative of His in which the alpha atoms aremodified. The His derivative in exemplary aspects is D-histidine,desaminohistidine, hydroxyl-histidine, acetyl-histidine, homo-histidine,N-methyl histidine, alpha-methyl histidine, imidazole acetic acid, oralpha, alpha-dimethyl imidiazole acetic acid (DMIA).

In exemplary aspects, the amino acid at position 1 comprises an analogstructure of Formula A, wherein one or both of the ring nitrogens areattached to a C1-C3 alkyl.

In yet other aspects, the amino acid at position 1 is a DPP-IVprotective amino acid, as described herein. In some aspects, the DPP-IVprotective amino acid is a derivative of His.

Position 2

In some embodiments, the presently disclosed peptides comprise a DPP-IVprotective amino acid at position 2. As used herein, the term “DPP-IVprotective amino acid” refers to an amino acid which achievessubstantial resistance of the presently disclosed peptide againstdipeptidyl peptidase IV (DPP IV) cleavage. In some aspects, the DPP-IVprotective amino acid is one of D-serine, D-alanine, valine, glycine,N-methyl serine, N-methyl alanine, or alpha, aminoisobutyric acid (AIB).In some aspects, the DPP-IV protective amino acid is anα,α-disubstituted amino acid. In some aspects, the α,α-disubstitutedamino acid comprises R1 and R2, each of which is bonded to the alphacarbon, wherein each of R1 and R2 is independently selected from thegroup consisting of C1-C4 alkyl, optionally substituted with a hydroxyl,amide, thiol, halo, or R1 and R2 together with the alpha carbon to whichthey are attached form a ring. In some aspects, the α,α-disubstitutedamino acid is AIB or 1-aminocyclopropane-1-carboxylate (ACPC).

The term “C₁-C_(n) alkyl” wherein n can be from 1 through 6, as usedherein, represents a branched or linear alkyl group having from one tothe specified number of carbon atoms. Typical C₁-C₆ alkyl groupsinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

In exemplary embodiments, the DPP-IV protective amino acid is theD-isomer of Ser (D-Ser), or a conservative amino acid substitutionthereof. For example, the DPP-IV protective amino acid can comprise aside chain structure of —(C1-C4 alkyl)OH. Optionally, when the sidechain structure comprises —(C3 alkyl)OH or —(C4 alkyl)OH, the carbonchain may be straight chained or branched.

In exemplary aspects, when the DPP-IV protective amino acid is D-Ser andthe amino acid at position 1 is His, the GIP agonist peptide is notconjugated to a heterologous moiety, e.g., a hydrophilic moiety (e.g.,PEG). In other aspects of the present disclosures, the DPP-IV protectiveamino acid is not D-serine.

Position 3

In some embodiments, the glucagon analog comprises at position 3 anamino acid which confers to the glucagon analog a reduced activity atthe glucagon receptor, as compared to a matched control peptide havingthe native glucagon residue at this position (Gln). In some aspects, theglucagon analogs having this type of amino acid at position 3 have about10% or less of the activity of native glucagon at the glucagon receptor,e.g., about 1-10%, or about 0.1-10%, or greater than about 0.1% but lessthan about 10%. In some embodiments, the glucagon analogs exhibit about0.5%, about 1% or about 7% of the activity of native glucagon. Inexemplary aspects, the amino acid at position 3 is an acidic, basic, orhydrophobic amino acid residue, e.g., glutamic acid, ornithine,norleucine. In some aspects, the amino acid at position 3 is Ile or Leu.

In some embodiments, the glucagon analog comprises the native amino acidof SEQ ID NO: 1 at position 3, e.g., glutamine, or comprises a glutamineanalog. Without being bound to a particular theory, such glucagonanalogs comprising a glutamine analog do not exhibit a substantial lossof activity at the glucagon receptor, and in some cases, the glucagonanalog comprising the glutamine analog enhances glucagon receptoractivity. In some embodiments, the glutamine analog comprises atposition 3 an amino acid comprising a side chain of Structure I, II orIII:

wherein R¹ is C₀₋₃ alkyl or C₀₋₃ heteroalkyl; R² is NHR⁴ or C₁₋₃ alkyl;R³ is C₁₋₃alkyl; R⁴ is H or C₁₋₃ alkyl; X is NH, O, or S; and Y is NHR⁴,SR³, or OR³. In some embodiments, X is NH or Y is NHR⁴. In someembodiments, R¹ is C₀₋₂ alkyl or C₁ heteroalkyl. In some embodiments, R²is NHR⁴ or C₁ alkyl. In some embodiments, R⁴ is H or C¹ alkyl. Inexemplary embodiments, an amino acid comprising a side chain ofStructure I is provided where, R¹ is CH₂—S, X is NH, and R² is CH₃(acetamidomethyl-cysteine, C(Acm)); R¹ is CH₂, X is NH, and R² is CH₃(acetyldiaminobutanoic acid, Dab(Ac)); R¹ is C₀ alkyl, X is NH, R² isNHR⁴, and R⁴ is H (carbamoyldiaminopropanoic acid, Dap(urea)); R¹ isCH₂—CH₂, X is NH, and R² is CH₃ (acetylornithine, Orn(Ac)); or R¹ is(CH₂)₃, X is NH, and R² is CH₃ (acetyl lysine (Ac Lys). In exemplaryembodiments, In exemplary embodiments, an amino acid comprising a sidechain of Structure II is provide where, R¹ is CH₂, Y is NHR⁴, and R⁴ isCH₃ (methylglutamine, Q(Me)); In exemplary embodiments, an amino acidcomprising a side chain of Structure III is provided where, R¹ is CH₂and R⁴ is H (methionine-sulfoxide, M(O)); In specific embodiments, theamino acid at position 3 is substituted with Dab(Ac).

In some embodiments, the glucagon analog comprises at position 3:

-   -   (i) an aliphatic amino acid, e.g., Ala, Val, norleucine (Nle),        or norvaline (NorVal);    -   (ii) an amino acid with a side chain of structure S1:

—(CH₂)n-X  [Structure S1]

-   -   -   wherein n is an integer between 1 to 4; and        -   wherein X is SCH₃, S(O)CH₃; or S(O)₂; or

    -   (iii) an amino acid with a side chain of structure S2:

—(CH₂)n-OH  [Structure S2]

-   -   -   wherein n is an integer between 1 to 4.

In exemplary aspects, the amino acid with a side chain of structure S1is Met, methionine sulfoxide, or methionine sulfone. In exemplaryaspects, the amino acid with a side chain of structure S2 is homoserine.Without being bound to a particular theory, such glucagon analogsmaintain some glucagon activity.

Position 7

In some embodiments, the glucagon analog comprises an amino acidmodification at position 7, relative to SEQ ID NO: 1, e.g., the glucagonanalog comprises an amino acid other than Thr at position 7. In someaspects, the amino acid at position 7 is a large, aliphatic amino acid,e.g., Ile, Leu, Ala, and the like. Without being bound to a particulartheory, glucagon analogs comprising such an amino acid at position 7 arebelieved to exhibit drastically reduced activity at the GLP-1 receptor.

Position 9

In some embodiments, the glucagon analog comprises an amino acidmodification at position 9, relative to SEQ ID NO: 1, e.g., the glucagonanalog comprises an amino acid other than Asp at position 9. In someembodiments, the glucagon analog comprises a negative charged amino acidother than Asp, e.g., Glu, homoglutamic acid, cysteic acid, homocysteicacid. In some aspects, the amino acid at position 9 is an acylated aminoacid or an alkylated amino acid, as discussed herein.

Position 10

In some embodiments, the glucagon analog comprises an amino acidmodification at position 10, relative to SEQ ID NO: 1, e.g., theglucagon analog comprises an amino acid other than Tyr at position 10.In some aspects, the amino acid at position 10 is Trp. Without beingbound to a particular theory, glucagon analogs comprising such an aminoacid at position 10 are believed to exhibit activity at the GIPreceptor, GLP-1 receptor, and/or the glucagon receptor which is notreduced, as compared to the corresponding peptide with a Tyr at position10.

In some embodiments, the glucagon analog comprises an acylated aminoacid or an alkylated amino acid, as discussed herein.

Position 11

In some embodiments, the glucagon analog comprises an amino acidmodification at position 11, relative to SEQ ID NO: 1, e.g., theglucagon analog comprises an amino acid other than Ser at position 11.In some aspects, the amino acid at position 11 is a small aliphaticamino acid, e.g., Gly or Ala. Without being bound to a particulartheory, glucagon analogs comprising such an amino acid at position 10are believed to exhibit a reduced activity at the glucagon receptor.

Position 12

In some embodiments, the glucagon analog comprises an amino acidmodification at position 12, relative to SEQ ID NO: 1. e.g., theglucagon analog comprises an amino acid other than Lys at position 10.In some aspects, the amino acid at position 12 is a large, aliphatic,nonpolar amino acid, optionally, isoleucine. In some aspects, the aminoacid at position 12 is Arg. Without being bound to a particular theory,glucagon analogs comprising a large, aliphatic, nonpolar amino acid,e.g., Ile, exhibit enhanced activity at the GIP receptor. In someembodiments, the glucagon analog comprises an acylated amino acid or analkylated amino acid, as discussed herein.

Position 15

In some embodiments, the glucagon analogs comprise an amino acidmodification at position 15, relative to SEQ ID NO: 1, e.g., theglucagon analog comprises an amino acid other than Asp at position 15.In some aspects, the amino acid at position 15 is deleted or is glutamicacid, homoglutamic acid, cysteic acid or homocysteic acid. Without beingbound to a particular theory, such glucagon analogs exhibit improvedstability, e.g., by way of reducing degradation or cleavage of theanalog over time, especially in acidic or alkaline buffers, e.g.,buffers at a pH within the range of 5.5 to 8. In some embodiments, theglucagon analogs comprising this modification retains at least 75%, 80%,90%, 95%, 96%, 97%, 98% or 99% of the original analog after 24 hours at25° C.

Position 16

In some embodiments, the glucagon analogs comprise an amino acidmodification at position 16, relative to SEQ ID NO: 1, e.g., theglucagon analog comprises an amino acid other than Ser at position 16.In some aspects, the amino acid at position 16 is glutamic acid or withanother negative-charged amino acid having a side chain with a length of4 atoms, or alternatively with any one of glutamine, homoglutamic acid,or homocysteic acid, or a charged amino acid having a side chaincontaining at least one heteroatom, (e.g., N, O, S, P) and with a sidechain length of about 4 (or 3-5) atoms. In some embodiments, theglucagon analog comprises at position 16 an amino acid selected from thegroup consisting of glutamic acid, glutamine, homoglutamic acid,homocysteic acid, threonine or glycine or is an amino acid selected fromthe group consisting of glutamic acid, glutamine, homoglutamic acid andhomocysteic acid.

Without being bound to a particular theory, such glucagon analogsexhibit enhanced stability, e.g., by way of reducing degradation of thepeptide over time, especially in acidic or alkaline buffers, e.g.,buffers at a pH within the range of 5.5 to 8. Such glucagon analogs areless susceptible to cleavage of the Asp15-Ser16 peptide bond.

In some embodiments, the amino acid at position 16 is a negativelycharged amino acid, optionally in combination with an alpha helixpromoting amino acid (e.g., an alpha, alpha disubstituted amino acid, orAIB) at position 20. Such glucagon analogs exhibit GIP activity.

In alternative embodiments, the glucagon analog comprises at position 16a Thr or an alpha helix promoting amino acid, as described above. Insome embodiments, the alpha helix promoting amino acid is AIB or Glu.

In some aspects, the amino acid at position 16 is a positive chargedamino acid, e.g., Formula IV:

wherein n is 1 to 16, or 1 to 10, or 1 to 7, or 1 to 6, or 2 to 6, or 2or 3 or 4 or 5, each of R₁ and R₂ is independently selected from thegroup consisting of H, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄alkyl) (C₂-C₅ heterocyclic), (C₀-C₄ alkyl) (C₆-C₁₀ aryl)R₇, and (C₁-C₄alkyl) (C₃-C₉ heteroaryl), wherein R₇ is H or OH. In exemplary aspects,the amino acid at position 16 of the glucagon analog is Lys.

In some embodiments, the glucagon analog comprises an acylated aminoacid or an alkylated amino acid at position 16, as discussed herein.

In yet additional embodiments, the amino acid at position 16 is an aminoacid comprising a side chain which is conjugated to a heterologousmoiety, as described herein under the section entitled “CONJUGATES.”

In some embodiments, the glucagon analog comprises an aliphatic aminoacid at position 16. For example, the glucagon analogs may have a Gly,Ala, Val, Leu, or Ile, at position 16 of the glucagon analog. Inexemplary aspects, the glucagon analog comprises a small aliphaticresidue, e.g., Gly, Ala.

In exemplary aspects, the glucagon analog comprises an amino acidcomprising an imidazole side chain. In exemplary aspects, the amino acidat position 16 comprises a structure of Formula A:

wherein each of R¹ and R² independently is selected from the groupconsisting of H, (C1-6)alkyl, O(C1-6)alkyl, (C1-6)alkyl-OH, F, and(C1-C6)alkyl of which at least one H is replaced by F. In exemplaryaspects, the amino acid at position 16 comprises an analog structure ofFormula A, wherein one or both of the ring nitrogens are attached to aC1-C3 alkyl.

In exemplary aspects, the amino acid at position 16 is L-histidine(His). In other aspects, the amino acid at position 16 is a derivativeof His (His derivative), some of which are described herein. See, e.g.,the teachings under “Position 1.”

Positions 17 and 18

In some embodiments, the glucagon analog comprises an amino acidmodification at either or both of positions 17 and 18, relative to SEQID NO: 1, such that the dibasic Arg-Arg site at positions 17 and 18 iseliminated. In some embodiments, the glucagon analog comprises an aminoacid other than Arg at one or both of positions 17 and 18. Without beingbound to any particular theory, it is believed that elimination of thedibasic site improves the in vivo efficacy of the glucagon analog. Insome aspects, the amino acid at position 17 is not a basic amino acid.In some aspects, the amino acid at position 17 is an aliphatic aminoacid. In some embodiments, the amino acid at position 17 is substitutedwith another amino acid as described herein, e.g., an amino acidcomprising a hydrophilic moiety, an alpha helix promoting amino acid. Insome embodiments, the alpha helix promoting amino acid forms anon-covalent intramolecular bridge with an amino acid at j+3 or i+4. Insome aspects, the amino acid at position 17 is Gln.

In other aspects, the glucagon analog of the present disclosurescomprises an amino acid comprising an imidazole side chain at position17. In exemplary aspects, the amino acid at position 17 comprises astructure of Formula A:

wherein each of R¹ and R² independently is selected from the groupconsisting of H, (C1-6)alkyl, O(C1-6)alkyl, (C1-6)alkyl-OH, F, and(C1-C6)alkyl of which at least one H is replaced by F. In exemplaryaspects, the amino acid at position 17 comprises an analog structure ofFormula A, wherein one or both of the ring nitrogens are attached to aC1-C3 alkyl.

In exemplary aspects, the amino acid at position 17 is L-histidine(His). In other aspects, the amino acid at position 17 is a derivativeof His (His derivative), some of which are described herein. See, e.g.,the teachings under “Position 1.”

In some aspects, the amino acid at position 18 is not a basic aminoacid. In some aspects, the amino acid at position 18 is an aliphaticamino acid. In some embodiments, the amino acid at position 18 is asmall aliphatic amino acid, e.g., Ala.

In some specific aspects, the amino acid at position 18 is a smallaliphatic amino acid, e.g., Ala, and the amino acid at position 17 isArg. In other aspects, the amino acid at position 18 is a smallaliphatic amino acid, e.g., Ala, and amino acid at position 17 is Gln.

In some aspects, the amino acid at position 17 is an amino acidcomprising a side chain which is conjugated to a heterologous moiety, asdescribed herein under the section entitled “CONJUGATES.

Position 19

In some embodiments, the glucagon analogs comprise an amino acidmodification at position 19, relative to SEQ ID NO: 1, e.g., theglucagon analog comprises an amino acid other than Ala at position 19.In some aspects, the amino acid at position 19 is an alpha helixpromoting amino acid (e.g., an alpha, alpha disubstituted amino acid, orAIB). Without being bound to any particular theory, a glucagon analogcomprising an alpha, alpha disubstituted amino acid, e.g., an AIB,exhibits reduced glucagon agonist activity, as compared to a matchedcontrol peptide, wherein the amino acid at position 19 is the nativeamino acid residue, Ala.

Position 20

In some embodiments, the glucagon analog comprises an amino acidmodification at position 20, relative to SEQ ID NO: 1, e.g., the aminoacid at position 20 is an amino acid other than Gln. In some aspects,the amino acid at position 20 is an alpha helix promoting amino acid,e.g. as described above. In some aspects, the amino acid at position 20is an alpha, alpha disubstituted amino acid, e.g., AIB, ACPC. In someembodiments, the alpha helix promoting amino acid forms a non-covalentintramolecular bridge with an amino acid at j−3 or i−4.

In some specific embodiments the amino acid is a hydrophilic amino acidhaving a side chain that is either charged or has an ability tohydrogen-bond, and is at least about 5 (or about 4-6) atoms in length,for example, lysine, citrulline, arginine, or ornithine. In otheraspects, the amino acid at position 20 is Ser, Thr, Ala or AIB.

In some aspects, the amino acid at position 20 is an acylated amino acidor alkylated amino acid, as discussed herein.

In some aspects, the amino acid at position 20 is an amino acidcomprising a side chain which is conjugated to a heterologous moiety, asdescribed herein under the section entitled “CONJUGATES.

In some aspects, the amino acid at position 20 of the glucagon analogcomprises an imidazole side chain. In exemplary aspects, the amino acidat position 20 comprises a structure of Formula A:

wherein each of R1 and R2 independently is selected from the groupconsisting of H, (C1-6)alkyl, O(C1-6)alkyl, (C1-6)alkyl-OH, F, and(C1-C6)alkyl of which at least one H is replaced by F. In exemplaryaspects, the amino acid at position 20 comprises an analog structure ofFormula A, wherein one or both of the ring nitrogens are attached to aC1-C3 alkyl.

In exemplary aspects, the amino acid at position 20 is L-histidine(His). In other aspects, the amino acid at position 20 is a derivativeof His (His derivative), some of which are described herein. See, e.g.,the teachings under “Position 1.”

In some aspects, the amino acid at position 20 is a positive chargedamino acid, e.g., Formula IV:

wherein n is 1 to 16, or 1 to 10, or 1 to 7, or 1 to 6, or 2 to 6, or 2or 3 or 4 or 5, each of R₁ and R₂ is independently selected from thegroup consisting of H, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄alkyl)(C₃-C₉ heteroaryl), wherein R₇ is H or OH. In exemplary aspects,the amino acid at position 20 of the glucagon analog is Lys.

Without being bound to a particular theory, such glucagon analogsexhibit enhanced activity at the GLP-1 receptor and/or GIP receptor orexhibit reduced degradation that occurs through deamidation of Glnand/or exhibit increased stability.

Position 21

In some embodiments, the glucagon analog comprises an amino acidmodification at position 21, relative to SEQ ID NO: 1, e.g., the aminoacid at position 21 is an amino acid other than Asp. In exemplaryaspects, the amino acid at position 21 is Ser, Thr, Ala or AIB. In otheraspects, the amino acid at position 21 is Lys, Arg, Orn, or Citrulline.In some aspects, the amino acid at position 21 is Glu, homoglutamic acidor homocysteic acid. In some aspects, the amino acid at position 21 isan amino acid comprising a side chain which is conjugated to aheterologous moiety, as described herein under the section entitled“CONJUGATES.

In some embodiments, the amino acid at position 21 is an alpha helixpromoting amino acid. In some embodiments, the alpha helix promotingamino acid forms a non-covalent intramolecular bridge with an amino acidat j−3 or i−4.

Without being bound to a particular theory, such glucagon analogsexhibit reduced degradation that occurs through degradation throughdehydration of Asp to form a cyclic succinimide intermediate followed byisomerization to iso-aspartate and/or exhibit increased stability.

Position 23

In some aspects, the glucagon analog comprises an amino acidmodification at position 23, relative to SEQ ID NO: 1. In some aspects,the amino acid at position 23 is an amino acid other than Val, includingbut not limited to Ile.

Position 24

In some aspects, the glucagon analog comprises an amino acidmodification at position 24, relative to SEQ ID NO: 1. In some aspects,the amino acid at position 24 is an amino acid other than Gln, e.g.,Ala, Asn, Cys. In some aspects, the amino acid at position 24 is anamino acid comprising a side chain which is conjugated to a heterologousmoiety, as described herein under the section entitled “CONJUGATES.

Position 27

In some aspects, the glucagon analog comprises an amino acidmodification at position 27, relative to SEQ ID NO: 1. In some aspects,the amino acid at position 27 is an amino acid other than Met. In someembodiments, the glucagon analog comprises at position 27 an amino acidwhich prevents oxidative degradation of the peptide. In some aspects,the amino acid at position 27 is methionine sulfoxide, leucine,isoleucine or norleucine. In some specific embodiments, the amino acidat position 27 is leucine or norleucine.

In other aspects, the amino acid at position 27 is an aliphatic aminoacid (e.g., Gly, Ala, Val, Leu, Ile) or an amino acid of Formula IV, asdescribed herein, e.g., Lys. In exemplary embodiments, the amino acid atposition 27 is Val or Lys. Without being bound to any particular theory,such an amino acid modification reduces glucagon activity.

Positions 28 and 29

In some aspects, the glucagon analog comprises an amino acidmodification at position 28, relative to SEQ ID NO: 1. In some aspects,the amino acid at position 28 is an amino acid other than Asn. In someaspects, the amino acid at position 28 is Ala, Ser, Thr, or AIB. In someaspects, the amino acid at position 28 is a charged amino acid, e.g., anegative-charged amino acid, as further described herein. See sectionentitled “Charged C-terminus.” In some aspects, the amino acid atposition 28 is Asp.

In exemplary aspects, the amino acid at position 28 is an amino acid ofFormula IV as described herein. The amino acid in exemplary embodimentsis Lys. Without being bound to any particular theory, such an amino acidmodification reduces glucagon activity.

In some aspects, the glucagon analog comprises an amino acidmodification at position 29, relative to SEQ ID NO: 1. In some aspects,the amino acid at position 29 is an amino acid other than Thr. In someaspects, the amino acid at position 29 is an aliphatic residue, e.g.,Gly, Ala, Val, Leu, Ile. In some aspects, the amino acid at position 29is a small aliphatic amino acid. In some aspects, the amino acid atposition 29 is Ala or Gly. In some aspects, the amino acid at position29 is an α,α-disubstituted amino acid, such as any of these describedherein. In exemplary aspects, the amino acid at position 29 is AIB.

In some aspects, the amino acid at position 29 is an amino acidcomprising a side chain which is conjugated to a heterologous moiety, asdescribed herein under the section entitled “CONJUGATES.

Without being bound to any particular theory, when position 28 is Aspand position 29 is Gly, the glucagon analog is subject to a degree ofchemical instability. Thus, in exemplary aspects, the glucagon analogdoes not have both Asp at position 28 and Gly at position 29, and mayexhibit enhanced chemical stability. For example, in some aspects, theglucagon analog comprises Asp at position 28 and an amino acid otherthan Gly at position 29. In exemplary aspects, the glucagon analogcomprises Asp at position 28 and one of Gly, Ala, Val, Leu, Ile, or AIBat position 29. In alternative aspects, the glucagon analog comprisesGly at position 29 and an amino acid other than Asp at position 28. Inexemplary aspects, the glucagon analog comprises Ala, Ser, Thr, Lys,AIB, or Glu at position 28 and Gly at position 29. In exemplary aspects,the glucagon analog comprises Glu at position 28 and Gly at position 29.

Charged C-Terminus

In some embodiments, the glucagon analog comprises one or more aminoacid substitutions and/or additions that introduce a charged amino acidinto the C-terminal portion of the analog, relative to SEQ ID NO: 1. Insome embodiments, such modifications enhance stability and solubility.As used herein the term “charged amino acid” or “charged residue” refersto an amino acid that comprises a side chain that is positive-charged ornegative-charged (i.e., de-protonated) or positive-charged (i.e.,protonated) in aqueous solution at physiological pH. In some aspects,these amino acid substitutions and/or additions that introduce a chargedamino acid modifications are at a position C-terminal to position 27 ofSEQ ID NO: 1. In some embodiments, one, two or three (and in someinstances, more than three) charged amino acids are introduced withinthe C-terminal portion (e.g., position(s) C-terminal to position 27). Inaccordance with some embodiments, the native amino acid(s) at positions28 and/or 29 are substituted with a charged amino acids, and/or in afurther embodiment one to three charged amino acids are also added tothe C-terminus of the analog. In exemplary embodiments, one, two or allof the charged amino acids are negative-charged. The negative-chargedamino acid in some embodiments is aspartic acid, glutamic acid, cysteicacid, homocysteic acid, or homoglutamic acid. In some aspects, thesemodifications increase solubility or stability. In some embodiments,position 30 is not a charged amino acid. Without being bound to aparticular theory, a charged amino acid, e.g., a negative charged aminoacid, e.g., Glu, reduced GIP activity.

C-Terminal Truncation

In accordance with some embodiments, the glucagon analogs disclosedherein are modified by truncation of the C-terminus by one or two aminoacid residues. Such modified glucagon peptides, retain similar activityand potency at the glucagon receptor and GLP-1 receptor. In this regard,the glucagon peptides can comprise amino acids 1-27 or 1-28 of thenative glucagon analog (SEQ ID NO: 1), optionally with any of theadditional modifications described herein.

Charge-Neutral C-Terminus

In some embodiments, the glucagon analog comprises a charge-neutralgroup, such as an amide or ester, at the C-terminus in place of thealpha carboxylate, relative to SEQ ID NO: 1. Without being bound to anyparticular theory, such modifications in exemplary aspects increasesactivity of the glucagon analog at the GLP-1 receptor. Accordingly, insome embodiments, the glucagon analog is an amidated peptide, such thatthe C-terminal residue comprises an amide in place of the alphacarboxylate of an amino acid. As used herein a general reference to apeptide or analog is intended to encompass peptides that have a modifiedamino terminus, carboxy terminus, or both amino and carboxy termini. Forexample, an amino acid chain composing an amide group in place of theterminal carboxylic acid is intended to be encompassed by an amino acidsequence designating the standard amino acids.

C-Terminal Extensions

In some embodiments of the present disclosures, the glucagon analogscomprise a C-terminal extension of 1-21 amino acids fused to the aminoacid at position 29. The C-terminal extension may comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 aminoacids. In some aspects, the C-terminal extension is any of theheterologous peptides described below in the section “CONJUGATES.” Forexample, the glucagon analog comprises at position 30 an aliphaticresidue, e.g., Gly, Ala, Val, Leu, Ile. In exemplary aspects, theglucagon analog comprises at position 30 a small aliphatic residue,e.g., Gly, Ala.

Also, for example, in some aspects, the extension comprises an aminoacid sequence which forms a Trp cage structure, e.g., the extensioncomprises the amino acid sequence of GPSSGAPPPS (SEQ ID NO: 5), or aconservatively substituted sequence thereof. In alternative aspects, theextension of 1 to 21 amino acids comprises at least one charged aminoacid. In exemplary aspects, the extension comprises an amino acidsequence of: X1-X2, wherein X1 is a charged amino acid and X2 is a smallaliphatic amino acid. In some aspects, X1 is a positive charged aminoacid, e.g., Arg. In some aspects, extension comprises Arg-Gly.

In some embodiments, the extension comprises an amino acid sequence ofSEQ ID NO: 5 (GPSSGAPPPS), SEQ ID NO: 6 (GGPSSGAPPPS), SEQ ID NO: 7(KRNRNNIA), or SEQ ID NO: 8 (KRNR). In specific aspects, the amino acidsequence is attached through the C-terminal amino acid of the glucagonanalog, e.g., amino acid at position 29. In some embodiments, the aminoacid sequence of any of SEQ ID NOs: 5-8 is bound to amino acid 29 of theglucagon analog through a peptide bond. In some specific embodiments,the amino acid at position 29 of the glucagon analog is a Gly and theGly is fused to one of the amino acid sequences of any of SEQ ID NOs:5-8.

In exemplary aspects, the glucagon analog comprises an extension whichforms a forms a structure known in the art as a Trp cage (see, e.g.,Paschek et al., Proc Natl Acad Sci USA 105 (46): 17754-17759 (2008). Insome aspects, the extension comprises the amino acid sequence GPSSGAPPPS(SEQ ID NO: 5) or GGPSSGAPPPS (SEQ ID NO: 6) or GPSSGRPPPS (SEQ ID NO:183) or a sequence of one of the foregoing with 1, 2, or 3 conservativeamino acid substitutions. In exemplary aspects, when the extensioncomprises the amino acid sequence of SEQ ID NO: 183, the amino acid atposition 28 is a negative charged amino acid, e.g., Asp or Glu.

Other Modifications

Descriptions of yet other modifications, relative to SEQ ID NO: 1, ofthe glucagon analogs of the present disclosures are found throughoutthis application. The above listing is not exhaustive, but merelyexemplary.

In some embodiments, the glucagon analogs described herein areglycosylated, amidated, carboxylated, phosphorylated, esterified,N-acylated, cyclized via, e.g., a disulfide bridge, or converted into asalt (e.g., an acid addition salt, a basic addition salt), and/oroptionally dimerized, multimerized, or polymerized, or conjugated.

Excluded Peptides

The glucagon analogs of the present disclosures are structurallydistinct from the glucagon analogs which exhibit GIP receptor agonistactivity described in International Patent Application No. PCTUS2009/47447 (filed on Jun. 16, 2009), U.S. Application No. 61/073,274(filed Jun. 17, 2008); U.S. Application No. 61/078,171 (filed Jul. 3,2008); U.S. Application No. 61/090,448 (filed Aug. 20, 2008), U.S.Application No. 61/151,349 (filed Feb. 10, 2009), U.S. Application No.61/187,578 (filed Jun. 16, 2009), International Patent Application No.PCT/US2010-038825 (filed Jun. 16, 2010); U.S. Provisional PatentApplication No. 61/426,285, filed on Dec. 22, 2010; U.S. ProvisionalPatent Application No. 61/514,609, filed on Aug. 3, 2011, andInternational Patent Application No. PCT/US2011/066164, filed Dec. 20,2011; the contents of which are incorporated by reference in theirentirety. Accordingly, in any or all embodiments, the glucagon analog ofthe present disclosures is not any of the glucagon analogs or peptidesdescribed in International Patent Application No. PCT/US2009/47447(filed on Jun. 16, 2009, and published as WO 2010/011439), U.S.Application No. 61/073,274 (filed Jun. 17, 2008); U.S. Application No.61/078,171 (filed Jul. 3, 2008); U.S. Application No. 61/090,448 (filedAug. 20, 2008), U.S. Application No. 61/151,349 (filed Feb. 10, 2009),U.S. Application No. 61/187,578 (filed Jun. 16, 2009), InternationalPatent Application No. PCT/US2010/038825 (filed Jun. 16, 2010, andpublished as WO 2010/148089), U.S. Application No. 61/298,812 (filedJan. 27, 2010), International Patent Application No. PCT/US2011/022608(filed Jan. 26, 2011, and published as WO 2011/094337), U.S. ProvisionalPatent Application No. 61/426,285, filed on Dec. 22, 2010; U.S.Provisional Patent Application No. 61/514,609, filed on Aug. 3, 2011, orInternational Patent Application No. PCT/US2011/066164, filed Dec. 20,2011. In exemplary embodiments, the peptides, glucagon peptides, orglucagon analogs of the present disclosures is not (i.e., excludes) anyone or all of the peptides of SEQ ID NOs: 1-262 of WO 2010/011439; SEQID NOs: 1-680 of WO 2010/148089, or SEQ ID NOs: 1-1318 ofPCT/US2011/022608, which published as WO2011/094337, on Aug. 4, 2011, orSEQ ID NOs: 10-262 of International Patent Application No.PCT/US2011/066164, filed Dec. 20, 2011, which published as InternationalPatent Application Publication No. WO2012/088116, on Jun. 28, 2012.

In exemplary embodiments, the peptides, glucagon peptides, or glucagonanalogs of the present disclosures is not (i.e., excludes) any one orall of the peptides of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, and/or 262 of International PatentApplication Publication No. WO 2010/011439.

In exemplary embodiments, the peptides, glucagon peptides, or glucagonanalogs of the present disclosures is not (i.e., excludes) any one orall of the peptides of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521,522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535,536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549,550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563,564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577,578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591,592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605,606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619,620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633,634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647,648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661,662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675,676, 677, 678, 679, and/or 680 of International Patent ApplicationPublication No. WO 2010/148089. In exemplary embodiments, the peptides,glucagon peptides, or glucagon analogs of the present disclosures is not(i.e., excludes) any one or all of the peptides of SEQ ID NO: 657, 658,659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669 of InternationalPatent Application Publication No. WO 2010/148089, which are presentedherein as SEQ ID NOs: 219-229, respectively.

In exemplary embodiments, the peptides, glucagon peptides, or glucagonanalogs of the present disclosures is not (i.e., excludes) any one orall of the peptides of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506,507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534,535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548,549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562,563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576,577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590,591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604,605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618,619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632,633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646,647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660,661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674,675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688,689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702,703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716,717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730,731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744,745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758,759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772,773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786,787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800,801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814,815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828,829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842,843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856,857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870,871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884,885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898,899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912,913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926,927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940,941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954,955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968,969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982,983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996,997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008,1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020,1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032,1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044,1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056,1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068,1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080,1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092,1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104,1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116,1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128,1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140,1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152,1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164,1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176,1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188,1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200,1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212,1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224,1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236,1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248,1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260,1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272,1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284,1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296,1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308,1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, and/or 1318 ofInternational Application No. Publication No. WO/2011/094337.

Exemplary Embodiments

In exemplary embodiments, the peptide of the present disclosures is ananalog of glucagon (SEQ ID NO: 1) comprising (i) at position 1 an aminoacid comprising an imidazole side chain, (ii) at position 2 a DPP-IVprotective amino acid, (iii) at any of positions 9, 10, 12, 16, 20, or37-43 an acylated amino acid or alkylated amino acid, optionally,wherein the acyl or alkyl group is linked to the amino acid via aspacer; (iv) at position 20 an amino acid selected from the groupconsisting of: (i) an α,α-disubstituted amino acid; and (ii) an aminoacid comprising an imidazole side chain; and (v) up to ten (e.g., 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acid modificationsrelative to SEQ ID NO: 1, wherein, when the glucagon analog is notconjugated to a heterologous moiety, e.g., a hydrophilic moiety (e.g.,PEG), the glucagon analog exhibits at least or about 0.1% (e.g., atleast or about 1%, at least or about 10%, at least or about 50%, atleast about 80%, at least or about 100%, at least or about 500%)activity of native GIP at the GIP receptor.

The glucagon analogs described here may comprise any activity profiledescribed herein. See, e.g., the section entitled “ACTIVITY OF THEPRESENTLY DISCLOSED PEPTIDES.” In exemplary aspects, the glucagon analogexhibits a GIP percentage potency of at least or about 1%, at least orabout 10%, at least or about 50%, at least or about 90%, at least orabout 100%, at least or about 300%, or at least or about 500%. In someaspects, the glucagon analog also exhibits a GLP-1 percentage potency ofat least or about 1%, at least or about 10%, at least or about 50%, atleast or about 90%, at least or about 100%, at least or about 300%, orat least or about 500%. In alternative or additional aspects, theglucagon analog exhibits a glucagon percentage potency of at least orabout 1%, at least or about 10%, at least or about 50%, at least orabout 90%, or at least or about 100%. Accordingly, while the glucagonanalogs may be considered as GIP agonist peptides, in some aspects, theglucagon analogs additionally may be considered as a GIP-GLP-1co-agonist, a GIP-glucagon co-agonist, or a GIP-GLP-1-glucgaontriagonist. For example, the peptide may exhibit agonist activity ateach of the human GIP receptor, the human GLP-1 receptor and optionallythe human glucagon receptor, wherein the peptide exhibits an EC50 at theGIP receptor which is within 100-fold (e.g., 50-fold, 40-fold, 30-fold,20-fold, 15-fold, 10-fold, or less) of its EC50 at the GLP-1 receptorand optionally is within 100-fold (e.g., 50-fold, 40-fold, 30-fold,20-fold, 15-fold, 10-fold, or less) of its EC50 at the glucagonreceptor.

In exemplary embodiments, the glucagon analog comprises an amino acidcomprising an imidazole side chain at position 1. In exemplary aspects,the amino acid at position 1 comprises a structure of Formula A

wherein each of R1 and R2 independently is selected from the groupconsisting of H, (C1-6)alkyl, O(C16)alkyl, (C16)alkyl-OH, F, and(C1-C6)alkyl of which at least one H is replaced by F. In exemplaryaspects, the amino acid at position 1 comprises an analog structure ofFormula A, wherein one or both of the ring nitrogens are attached to aC1-C3 alkyl.

In exemplary aspects, the amino acid at position 1 is the native residueof glucagon (SEQ ID NO: 1) L-histidine (His), or is a derivative of His(His derivative), e.g., a derivative of His in which the alpha atoms aremodified. As used herein, the term “His derivative” refers to a chemicalmoiety comprising an imidazole, e.g., comprising a structure of FormulaA, or a substituted imidazole, attached to at least one carbon atom. Inexemplary embodiments, the His derivative comprises a structure similarto the structure of histidine, except that the alpha amine, alphacarbon, or alpha carboxylate is replaced with another chemical moiety.In exemplary embodiments, the His derivative is an alpha substitutedhistidine of which the hydrogen atom linked to the alpha carbon issubstituted with another chemical moiety, e.g., methyl, ethyl, propyl,isopropyl, hydroxyl, methoxy, ethoxy, and the like. The His derivativein some aspects is D-histidine, desaminohistidine, hydroxyl-histidine,acetyl-histidine, homo-histidine, N-methyl histidine, alpha-methylhistidine, imidazole acetic acid, or alpha, alpha-dimethyl imidiazoleacetic acid (DMIA).

In some aspects, the DPP-IV protective amino acid at position 2 is oneof D-serine, D-alanine, valine, glycine, N-methyl serine, N-methylalanine, or alpha, aminoisobutyric acid (AIB). In some aspects, theDPP-IV protective amino acid is D-Ser, or a conservative amino acidsubstitution thereof, or an α,α-disubstituted amino acid. In someaspects, the α,α-disubstituted amino acid comprises R1 and R2, each ofwhich is bonded to the alpha carbon, wherein each of R1 and R2 isindependently selected from the group consisting of C1-C4 alkyl,optionally substituted with a hydroxyl, amide, thiol, halo, or R1 and R2together with the alpha carbon to which they are attached form a ring.In some aspects, the α,α-disubstituted amino acid is AIB. In exemplaryaspects, when the DPP-IV protective amino acid is D-Ser, the GIP agonistpeptide is not conjugated to a heterologous moiety, e.g., a hydrophilicmoiety (e.g., PEG). In other aspects of the present disclosures, theDPP-IV protective amino acid is not D-serine.

In exemplary aspect, the analog of glucagon (SEQ ID NO: 1) of thepresent disclosures comprises at position 20 an α,α-disubstituted aminoacid. In exemplary aspects, the α,α-disubstituted amino acid comprisesR1 and R2, each of which is bonded to the alpha carbon, wherein each ofR1 and R2 is independently selected from the group consisting of C1-C4alkyl, optionally substituted with a hydroxyl, amide, thiol, halo, or R1and R2 together with the alpha carbon to which they are attached form aring. In some embodiments, the α,α-disubstituted amino acid is1-aminocyclopropane-1-carboxylate (ACPC). In some aspects, theα,α-disubstituted at position 20 is AIB. In exemplary embodiments, theamino acid at position 16 is a charged amino acid, e.g., apositive-charged amino acid, negative charged amino acid. In someaspects, when the amino acid at position 20 is an alpha, alphadisubstituted amino acid, the amino acid at position 16 is apositive-charged amino acid of Formula IV, e.g., Lys, or is anegative-charged amino acid, e.g., Glu. In some aspects, when the aminoacid at position 20 is an alpha, alpha disubstituted amino acid, theamino acid at position 16 is a charge neutral amino acid, e.g., Ser,Ala, Gly. In exemplary aspects, the amino acid at position 16 is a smallaliphatic amino acid (e.g., Ala), an α,α-disubstituted amino acid (e.g,AIB), or an amino acid comprising an imidazole side chain (e.g., His).In exemplary aspects, the glucagon analog comprises at position 17 ofthe glucagon analog an Arg, Gln, or an amino acid comprising animidazole side chain, optionally, His. In exemplary aspects, theglucagon analog comprises Ala at position 18 and Ala at position 19. Inexemplary aspects, the glucagon analog comprises Glu at position 21. Inexemplary aspects, the glucagon analog comprises a Leu or Nle atposition 27, an Asp or Glu at position 28, and a Gly at position 29. Inexemplary aspects, the glucagon analog comprises a C-terminal extensionof 1-21 amino acids C-terminal to the amino acid at position 29. Inexemplary aspects, the glucagon analog comprises a C-terminal amide inplace of the alpha carboxylate of the C-terminal residue.

In exemplary aspects, the analog of glucagon (SEQ ID NO: 1) of thepresent disclosures comprises at position 20 an amino acid comprising animidazole side chain. In exemplary aspects, the amino acid comprising animidazole side chain comprises a structure of Formula A:

wherein each of R1 and R2 independently is selected from the groupconsisting of H, (C1-6)alkyl, O(C1-6)alkyl, (C1-6)alkyl-OH, F, and(C1-C6)alkyl of which at least one H is replaced by F. Suitable aminoacids comprising an imidazole side chain are described herein. Inexemplary aspects, the amino acid at position 20 comprises an analogstructure of Formula A, wherein one or both of the ring nitrogens areattached to a C1-C3 alkyl. In some aspects, the amino acid at position20 is His or a His derivative. In exemplary aspects, the glucagon analogcomprises at position 16 a negative charged amino acid, e.g., Glu. Inexemplary aspects, the glucagon analog comprises at position 17 of theglucagon analog an Arg, Gln, or an amino acid comprising an imidazoleside chain, optionally, His. In exemplary aspects, the glucagon analogcomprises Ala at position 18 and Ala at position 19. In exemplaryaspects, the glucagon analog comprises a Leu or Nle at position 27, anAsp or Glu at position 28, and a Gly at position 29. In exemplaryaspects, the glucagon analog comprises a C-terminal extension of 1-21amino acids C-terminal to the amino acid at position 29. In exemplaryaspects, the glucagon analog comprises a C-terminal amide in place ofthe alpha carboxylate of the C-terminal residue.

In exemplary embodiments, the peptide of the present disclosures is ananalog of glucagon (SEQ ID NO: 1) comprising (i) an amino acid selectedfrom the group consisting of: Ala, Leu, Ile, Val, norvaline, homoserine,Met, and methionine sulfone, at position 3; (ii) at any of positions 9,10, 12, 16, 20, or 37-43 an acylated amino acid or alkylated amino acid,optionally, wherein the acyl or alkyl group is linked to the amino acidvia a spacer; (iii) an alpha helix stabilizing amino acid at one or moreof positions 16-21, and (v) up to ten (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8,9, or 10) additional amino acid modifications relative to SEQ ID NO: 1,wherein, when the glucagon analog is not conjugated to a heterologousmoiety, e.g., a hydrophilic moiety (e.g., PEG), the glucagon analogexhibits at least or about 0.1% (e.g., at least or about 1%, at least orabout 10%, at least or about 50%, at least about 80%, at least or about100%, at least or about 500%) activity of native GIP at the GIPreceptor.

The glucagon analogs comprising an amino acid modification which confersreduced activity at the glucagon receptor may comprise any activityprofile described herein. See, e.g., the section entitled “ACTIVITY OFTHE PRESENTLY DISCLOSED PEPTIDES.” In exemplary aspects, the glucagonanalog exhibits a GIP percentage potency of at least or about 1%, atleast or about 10%, at least or about 50%, at least or about 90%, atleast or about 100%, at least or about 300%, or at least or about 500%.In some aspects, the glucagon analog also exhibits a GLP-1 percentagepotency of at least or about 1%, at least or about 10%, at least orabout 50%, at least or about 90%, at least or about 100%, at least orabout 300%, or at least or about 500%. Accordingly, while the glucagonanalogs may be considered as GIP agonist peptides, in some aspects, theglucagon analogs additionally may be considered as a GIP-GLP-1co-agonist. For example, the peptide may exhibits agonist activity atthe human GIP receptor and the human GLP-1 receptor, wherein the peptideexhibits an EC50 at the GIP receptor which is within 100-fold (e.g.,50-fold, 40-fold, 30-fold, 20-fold, 15-fold, 10-fold, or less) of itsEC50 at the GLP-1 receptor.

In exemplary embodiments, the glucagon analog comprises an amino acidcomprising an imidazole side chain at position 1. In exemplary aspects,the amino acid at position 1 comprises a structure of Formula A

wherein each of R¹ and R² independently is selected from the groupconsisting of H, (C1-6)alkyl, O(C1-6)alkyl, (C1-6)alkyl-OH, F, and(C1-C6)alkyl of which at least one H is replaced by F. In exemplaryaspects, the amino acid at position 1 comprises an analog structure ofFormula A, wherein one or both of the ring nitrogens are attached to aC1-C3 alkyl.

In exemplary aspects, the amino acid at position 1 is the native residueof glucagon (SEQ ID NO: 1) L-histidine (His), or is a derivative of His(His derivative), e.g., a derivative of His in which the alpha atoms aremodified. As used herein, the term “His derivative” refers to a chemicalmoiety comprising an imidazole, e.g., comprising a structure of FormulaA, or a substituted imidazole, attached to at least one carbon atom. Inexemplary embodiments, the His derivative comprises a structure similarto the structure of histidine, except that the alpha amine, alphacarbon, or alpha carboxylate is replaced with another chemical moiety.In exemplary embodiments, the His derivative is an alpha substitutedhistidine of which the hydrogen atom linked to the alpha carbon issubstituted with another chemical moiety, e.g., methyl, ethyl, propyl,isopropyl, hydroxyl, methoxy, ethoxy, and the like. The His derivativein some aspects is D-histidine, desaminohistidine, hydroxyl-histidine,acetyl-histidine, homo-histidine, N-methyl histidine, alpha-methylhistidine, imidazole acetic acid, or alpha, alpha-dimethyl imidiazoleacetic acid (DMIA).

In some aspects, the glucagon analogs comprises a DPP-IV protectiveamino acid at position 2. In exemplary aspects, the DPP-IV protectiveamino acid is one of D-serine, D-alanine, valine, glycine, N-methylserine, N-methyl alanine, or alpha, aminoisobutyric acid (AIB). In someaspects, the DPP-IV protective amino acid is D-Ser, or a conservativeamino acid substitution thereof, or an α,α-disubstituted amino acid. Insome aspects, the α,α-disubstituted amino acid comprises R1 and R2, eachof which is bonded to the alpha carbon, wherein each of R1 and R2 isindependently selected from the group consisting of C1-C4 alkyl,optionally substituted with a hydroxyl, amide, thiol, halo, or R1 and R2together with the alpha carbon to which they are attached form a ring.In some aspects, the α,α-disubstituted amino acid is AIB. In exemplaryaspects, when the DPP-IV protective amino acid is D-Ser, the GIP agonistpeptide is not conjugated to a heterologous moiety, e.g., a hydrophilicmoiety (e.g., PEG). In other aspects of the present disclosures, theDPP-IV protective amino acid is not D-serine.

In exemplary aspects, the glucagon analog comprises an alpha helixstabilizing amino acid at one or more of positions 16-21. In exemplaryaspects, the glucagon analog comprises at position 16 an alpha helixstabilizing amino acid, optionally, a negative charged amino acid (e.g.,Glu) or an alpha, alpha disubstituted amino acid. In exemplary aspectsthe α,α-disubstituted amino acid comprises R1 and R2, each of which isbonded to the alpha carbon, wherein each of R1 and R2 is independentlyselected from the group consisting of C1-C4 alkyl, optionallysubstituted with a hydroxyl, amide, thiol, halo, or R1 and R2 togetherwith the alpha carbon to which they are attached form a ring. Inexemplary aspects, the α,α-disubstituted amino acid is AIB. In exemplaryaspects, the glucagon analog comprises an Arg at position 17 and a smallaliphatic amino acid, optionally, Ala, at position 18. In exemplaryaspects, the glucagon analog is not modified at position 20 compared toSEQ ID NO: 1 and the amino acid at position 20 is Gln. In exemplaryaspects, the glucagon analog comprises Glu at position 16, Arg atposition 17, Ala at position 18, Ala at position 19, and Gln at position20.

In exemplary aspects of the foregoing, the glucagon analog comprises anacylated or alkylated amino acid at any one of positions 9, 10, 12, 16,20. In exemplary aspects, the glucagon analog comprises an amino acidcovalently attached to a C12 to C18 acyl group or alkyl group at any oneor more of positions 9, 10, 12, 13, 14, 16, 17, and 20. In exemplaryaspects, the glucagon analog comprises an acylated or alkylated aminoacid at position 10. In some aspects, the glucagon analog comprises anextension of 1 to 21 amino acids C-terminal to the amino acid atposition 29 and comprises an acylated or alkylated amino acid at any ofpositions 37-43 (e.g., 37, 38, 39, 40, 41, 42, 43). In some aspects, theamino acid comprising a non-native acyl or alkyl group is at position40.

In exemplary aspects of the foregoing, the glucagon analog comprises anextension of 1 to 21 amino acids C-terminal to the amino acid atposition 29 and, in some aspects, the extension forms a structure knownin the art as a Trp cage. In some aspects, the extension comprises theamino acid sequence GPSSGAPPPS (SEQ ID NO: 5) or GGPSSGAPPPS (SEQ ID NO:6) or GPSSGRPPPS (SEQ ID NO:68) or a sequence of one of the foregoingwith 1, 2, or 3 conservative amino acid substitutions. In exemplaryaspects when the glucagon analog comprises an extension of 1 to 21 aminoacids, the glucagon analog comprises a Gly at position 29.

In exemplary aspects, the acylated amino acid or alkylated amino acidcomprises a structure of Formula I(optionally, Lys), Formula II,(optionally, Cys), or Formula III, (optionally, Ser). Optionally, insome aspects, the acylated amino acid or alkylated amino acid comprisesa structure of Formula I, e.g., Lys.

In some aspects, the acylated amino acid or alkylated amino acid islinked to the acyl group or alkyl group via a spacer. In some aspects,the spacer is 3 to 10 atoms in length. In some aspects, the spacer is anamino acid or dipeptide, and, in some aspects, the spacer comprises oneor two acidic amino acid residues, e.g., Glu. In some aspects, the acylor alkyl group is linked to the amino acid via wherein the total lengthof the spacer and the acyl group is about 14 to about 28 atoms inlength. In some aspects, the spacer comprises one or two gamma-Glu.

With regard to the acylated amino acid, the acyl group in some aspectsis a C12 to C18 (e.g., C12, C13, C14, C15, C16, C17, C18) fatty acylgroup. In some aspects, the acyl group is a C14 or C16 fatty acyl group.

In exemplary aspects of the foregoing embodiments, the glucagon analogcomprises a Glu at position 15, position 21, or position 28, or atpositions 15 and 21, at positions 15 and 28, or at positions 21 and 28,or at positions 15, 21, and 28.

In some aspects of the present disclosures, the glucagon analogcomprises at least one charged amino acid C-terminal to the amino acidat position 27. For example, in some aspects, the glucagon analogcomprises a charged amino acid (e.g., a negative charged amino acid) atposition 28. The negative charged amino acid in some aspects is Asp orGlu. The negative charged amino acid in some aspects is Glu. Inalternative aspects, the amino acid at position 28 is a positive chargedamino acid, e.g., a positive charged amino acid is an amino acid ofFormula I, e.g., Lys.

In alternative or additional aspects, the glucagon analog comprises anamino acid modification at position 27, at position 29, or at bothpositions 27 and 29. For example, the amino acid at position 27 is insome aspects is Leu, Nle, Val, or Lys and/or the amino acid at position29 is in some aspects Gly or Thr.

In some aspects, the amino acid at position 28 is Asp and optionally,the amino acid at position 29 is an amino acid other than Gly, e.g.,Val, Ala, or AIB.

The glucagon analogs described herein may comprise additional amino acidmodifications, e.g., up to ten (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)additional amino acid modifications, relative to SEQ ID NO: 1, asfurther discussed herein. In exemplary aspects, the glucagon analogcomprises one or more of:

-   -   i. an Ile at position 7;    -   ii. an Ile or Arg at position 12;    -   iii. an aliphatic amino acid, optionally, Ala, at position 18;    -   iv. an acidic amino acid, optionally, Glu, at position 21;    -   v. an Asn, Ala, or AIB at position 24;    -   vi. an aliphatic amino acid, optionally, Ala, or Leu, or Nle, at        position 27;    -   vii. an acidic amino acid, optionally, Glu, or an aliphatic        amino acid, optionally, Ala, at position 28;    -   viii. an aliphatic amino acid, optionally, Ala, at position 29;    -   ix. amidation at the C-terminus.

In accordance with the foregoing, the glucagon analog in exemplaryaspects comprises, consists essentially of, or consists of the aminoacid sequence of any of SEQ ID NOs: 19, 21-26, 28, 287, 289-294, 296,520, 522-527, and 529 or any of SEQ ID NOs: 30, 31, 32, 33, 35, 37, 38,41, 298, 299, 300, 301, 303, 305, 306, 309, 531, 532, 533, 534, 536,538, 539, and 542.

The invention provides a peptide comprising the sequence of SEQ ID NO:66,

(SEQ ID NO: 66)HX₂X₃GTFTSDX₁₀SKYLX₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁FVQWLX₂₇X₂₈X₂₉X₃₀PSSGX₃₅PPPSwherein:

-   -   X₂ is AIB;    -   X₃ is Gln or an amino acid comprising a side chain structure of        Structure I, II, or III;    -   X₁₀ is Tyr or an amino acid covalently attached to a C12 to C18        acyl or alkyl group;    -   X₁₅ is an acidic amino acid, optionally, Glu or Asp;    -   X₁₆ is any amino acid, optionally, any amino acid other than        Gly, Pro, and Ser; e.g., Glu, Ala, alpha, alpha-disubstituted        amino acid (e.g., AIB), His, Lys,    -   X₁₇ is Arg, His, Gln;    -   X₁₈ is Arg or Ala;    -   X₁₉ is Ala or alpha, alpha disubstituted amino acid (e.g., AIB);    -   X₂₀ is a selected from group consisting of: alpha,        alpha-disubstituted amino acid (e.g., AIB) or Gln or His, Lys,        or Ala;    -   X₂₁ is an acidic amino acid, optionally, Asp or Glu;    -   X₂₇ is Leu, Ala, Nle, or Met;    -   X₂₈ is Ala or an acidic amino acid (optionally, Asp or Glu);    -   X₂₉ is aliphatic, e.g., Ala or Gly or AIB or Val;    -   X₃₀ is small aliphatic amino acid, e.g., Ala or Gly    -   X₃₅ is Ala or a basic amino acid (optionally, Arg or Lys);    -   wherein, when X₂₈ is an acidic amino acid, X₃₅ is a basic amino        acid;    -   optionally, wherein, when X₂₈ is Asp, then X₂₉ is not Gly

wherein, when X₁₀ is Tyr, the peptide comprises at position 40 an aminoacid covalently attached to a C12 to C18 acyl or alkyl group, wherein,optionally, the peptide comprises Gly at position 41, and

wherein the C-terminal amino acid of the peptide is amidated.

In exemplary aspects of the peptide of SEQ ID NO: 66, X₁₀ is an aminoacid covalently attached to a C12 to C18 acyl or alkyl group. Inexemplary aspects, X₂₈ is Glu and optionally, X₁₅ is Glu and/or X₂₁ isGlu. In exemplary aspects, each of positions 15, 21, and 28. Inexemplary aspects, X₁₈ is Ala and X₁₉ is Ala and optionally, X₂₀ is analpha, alpha-disubstituted amino acid, e.g., AIB. In exemplary aspects,X₁₆ is Glu, Ala, AIB, His, or Lys; X₁₇ is Arg, His or Gln, and X₂₁ isGlu. In alternative exemplary aspects, X₂₀ is Lys or His and optionallyX₁₆ is Glu, X₁₇ is Arg, and X₂₁ is Asp. In alternative aspects, X₁₈ isAla and X₁₉ is an alpha, alpha-disubstituted amino acid (e.g., AIB) andoptionally, X₁₆ is Glu, X₁₇ is Arg, and X₂₁ is Glu. Also, in exemplaryaspects, X₂₀ is Ala or Lys. In exemplary aspects of the peptide of SEQID NO: 66, X₂₉ is Ala, Gly, Val, or an alpha, alpha-disubstituted aminoacid, optionally, AIB.

In exemplary aspects of the peptide of SEQ ID NO: 66, X₁₀ is Tyr and anamino acid covalently attached to a C12 to C18 acyl or alkyl group islocated at position 40. In exemplary aspects, the peptide comprises Glyat position 41. In exemplary aspects, X₉ or X₁₅ is Glu.

In exemplary aspects of the peptide of SEQ ID NO: 66, the acyl or alkylgroup is attached to an amino acid comprising a structure of Formula I,Formula II, or Formula III. In exemplary aspects, the amino acid ofFormula I is Lys. Also, in exemplary aspects, the acyl or alkyl group isattached to an amino acid of the peptide via a spacer, optionally aspacer that is an amino acid of dipeptide. In exemplary aspects, thespacer is one or two acidic amino acid residues.

In exemplary aspects of the peptide of SEQ ID NO: 66, X₃ is Gln oracetyl diaminobutanoic acid or acetyl Lys or acetyl ornithine or methylsulfoxide. In exemplary aspects, the peptide comprises Leu at position27, Gly at position 29, and GPSSGAPPPS C-terminal to Gly at position 29.

In exemplary aspects, the peptide comprises the sequence of any one ofSEQ ID NOs: 15-28, 34, 36, 39, 42, 43, 44, 52, 53, 63-65, 283-296, 302,304, 307, 310, 311, 312, 320, 321, 330-332, 516-529, 535, 537, 540, 543,544, 545, 553, 554, and 563-565.

The invention additionally provides a peptide comprising the sequenceSEQ ID NO: 66 with up to 3 (e.g., 1, 2, 3) amino acid modificationsrelative to SEQ ID NO: 66, wherein the peptide exhibits agonist activityat the human GIP receptor and, the human GLP-1 receptor and/or the humanglucagon receptor. The amino acid modifications may be any of thosedescribed herein. In exemplary aspects of the peptides comprising thesequence of SEQ ID NO: 66 with up to 3 (e.g., 1, 2, 3) amino acidmodifications relative to SEQ ID NO: 66, X₂₉ is not Gly when X₂₈ is Asp.In further exemplary aspects of the peptides comprising the sequence ofSEQ ID NO: 66 with up to 3 (e.g., 1, 2, 3) amino acid modificationsrelative to SEQ ID NO: 66, X₁ is an amino acid comprising a side chainimidazole, X₂ is a DPP-IV protective amino acid, optionally, an alpha,alpha-disubstituted amino acid, the C-terminal amino acid of the peptideis amidated, and the peptide comprises at position 40 an amino acidcovalently attached to a C12 to C18 acyl or alkyl group, when X₁₀ isTyr, and optionally, the peptide comprises Gly at position 41.

The invention also provides a peptide comprising the sequence of

-   -   (a) the sequence of

(SEQ ID NO: 67)X₁X₂X₃GTETSDX₁₀SKYLX₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁FVQWLX₂₇X₂₈X₂₉X₃₀PSSGX₃₅PPPSwherein:

-   -   X₁ is an amino acid comprising a side chain imidazole,        optionally, a His or His derivative;    -   X₂ is a DPP-IV protective amino acid, optionally, an alpha,        alpha-disubstituted amino acid (e.g., AIB);    -   X₃ is selected from the group consisting of Glu, Ala, Leu, Ile,        Nle, Val, NorVal, homoserine, Met, methionine sulfoxide,        methionine sulfone, acetyl-Orn, acetyl-diaminobutanoic acid,        acetyl-Lys;    -   X₁₀ is an amino acid covalently attached to a C12 to C18 acyl or        alkyl group;    -   X₁₅ is an acidic amino acid, optionally, Glu or Asp;    -   X₁₆ is any amino acid, optionally, any amino acid other than        Gly, Pro, and Ser; (e.g., Glu, Ala, alpha, alpha-disubstituted        amino acid (e.g., AIB), His, Lys)    -   X₁₇ is Arg, His, Gln;    -   X₁₈ is Arg or Ala;    -   X₁₉ is Ala or alpha, alpha disubstituted amino acid;    -   X₂₀ is a selected from group consisting of: alpha,        alpha-disubstituted amino acid (e.g., AIB) or Gln or His, Lys,        or Ala;    -   X₂₁ is an acidic amino acid, optionally, Asp or Glu;    -   X₂₇ is Leu, Ala, Nle, or Met;    -   X₂₈ is Ala or an acidic amino acid (optionally, Asp or Glu);    -   X₂₉ is aliphatic, e.g., Ala or Gly or AIB or Val;    -   X₃₀ is small aliphatic amino acid, e.g., Ala or Gly    -   X₃₅ is Ala or a basic amino acid (optionally, Arg or Lys);        wherein, when X₂₈ is an acidic amino acid, X₃₅ is a basic amino        acid;        optionally, wherein, when X₂₈ is Asp, then X₂₉ is not Gly        wherein, when X₁₀ is Tyr, the peptide comprises at position 40        an amino acid covalently attached to a C12 to C18 acyl or alkyl        group, and, wherein, optionally, the peptide comprises Gly at        position 41,        wherein the C-terminal amino acid of the peptide is amidated.

In exemplary aspects of the peptide comprising SEQ ID NO: 67, thepeptide comprises: X₁ is His; X₂ is AIB; X₁₆ is Glu; X₁₇ is Arg; X₁₈ isAla; X₁₉ is Ala; X₂₀ is Gln; X₂₇ is Leu; X₂₉ is Gly; X₃₀ is Gly; X₃₅ isAla; or a combination thereof. In exemplary aspects, the peptidecomprises the sequence of: SEQ ID NO: 29-42, 297-310, and 530-543.

The invention additionally provides a peptide comprising the sequenceSEQ ID NO: 67 with up to 3 (e.g., 1, 2, 3) amino acid modificationsrelative to SEQ ID NO: 67, wherein the peptide exhibits agonist activityat the human GIP receptor and, the human GLP-1 receptor and/or the humanglucagon receptor. The amino acid modifications may be any of thosedescribed herein. In exemplary aspects of the peptides comprising thesequence of SEQ ID NO: 67 with up to 3 (e.g., 1, 2, 3) amino acidmodifications relative to SEQ ID NO: 67, the amino acid at position 3 isselected from the group consisting of: Val, norvaline, homoserine, Met,methionine sulfoxide, methionine sulfone, acetyl-Orn,acetyl-diaminobutanoic acid, and acetyl Lys. In exemplary aspects of thepeptides comprising the sequence of SEQ ID NO: 67 with up to 3 (e.g., 1,2, 3) amino acid modifications relative to SEQ ID NO: 67, X₂₉ is not Glywhen X₂₈ is Asp. In further exemplary aspects of the peptides comprisingthe sequence of SEQ ID NO: 67 with up to 3 (e.g., 1, 2, 3) amino acidmodifications relative to SEQ ID NO: 67, X₁ is an amino acid comprisinga side chain imidazole, X₂ is a DPP-IV protective amino acid,optionally, an alpha, alpha-disubstituted amino acid, the C-terminalamino acid of the peptide is amidated, and the peptide comprises atposition 40 an amino acid covalently attached to a C12 to C18 acyl oralkyl group, when X₁₀ is Tyr, and optionally, the peptide comprises Glyat position 41. In exemplary aspects of the peptides comprising thesequence of SEQ ID NO: 67 with up to 3 amino acid modifications relativeto SEQ ID NO: 67, (i) X₁ is His; (ii) X₂ is AIB; (iii) X₁₆ is Glu; (iv)X₁₇ is Arg; (v) X₁₈ is Ala; (vi) X₁₉ is Ala; (vii) X₂₀ is Gln; (viii)X₂₇ is Leu; (ix) X₂₉ is Gly; (x) X₃₀ is Gly; (xi) X₃₅ is Ala; or (xii) acombination thereof.

The invention further provides a peptide comprising the sequence of SEQID NO: 18. In exemplary aspects, the peptide consists essentially of orconsists of SEQ ID NO: 18.

A peptide comprising the sequence of SEQ ID NO: 43 is also provided bythe invention. In exemplary aspects, the peptide consists essentially ofor consists of SEQ ID NO: 43.

Also provided herein is a peptide comprising a sequence of SEQ ID NO: 18with up to 3 (e.g., 1, 2, 3) amino acid modifications relative to SEQ IDNO: 18 or a sequence of SEQ ID NO: 43 with up to 3 (e.g., 1, 2, 3) aminoacid modifications relative to SEQ ID NO: 43, wherein the peptideexhibits agonist activity at the human GIP receptor and the human GLP-1receptor. In exemplary aspects of the peptides comprising the sequenceof SEQ ID NO: 18 or SEQ ID NO: 43 with up to 3 (e.g., 1, 2, 3) aminoacid modifications, the amino acid at position 29 is not Gly when theamino acid at position 28 is Asp. In further exemplary aspects of thepeptides comprising the sequence of SEQ ID NO: 18 or SEQ ID NO: 43 withup to 3 (e.g., 1, 2, 3) amino acid modifications, the amino acid atposition 1 is an amino acid comprising a side chain imidazole, the aminoacid at position 2 is a DPP-IV protective amino acid, optionally, analpha, alpha-disubstituted amino acid, the C-terminal amino acid of thepeptide is amidated, and the peptide comprises at position 40 an aminoacid covalently attached to a C12 to C18 acyl or alkyl group, when theamino acid at position 10 is Tyr, and optionally, the peptide comprisesGly at position 41. In exemplary aspects of the peptides comprising thesequence of SEQ ID NO: 18 with up to 3 (e.g., 1, 2, 3) amino acidmodifications or SEQ ID NO: 43 with up to 3 (e.g., 1, 2, 3) amino acidmodifications, (i) the amino acid at position 1 is His; (ii) the aminoacid at position 2 is AIB; (iii) the amino acid at position 16 is Glu;(iv) the amino acid at position 17 is Arg; (v) the amino acid atposition 18 is Ala; (vi) the amino acid at position 19 is Ala; (vii) theamino acid at position 20 is Gln; (viii) the amino acid at position 27is Leu; (ix) the amino acid at position 29 is Gly; (x) the amino acid atposition 20 is Gly; (xi) the amino acid at position 35 is Ala; or (xii)a combination thereof. In exemplary aspects of the peptides comprisingthe sequence of SEQ ID NO: 18 with up to 3 (e.g., 1, 2, 3) amino acidmodifications or SEQ ID NO: 43 with up to 3 (e.g., 1, 2, 3) amino acidmodifications, the amino acid at position 3 is selected from the groupconsisting of: Val, norvaline, homoserine, Met, methionine sulfoxide,methionine sulfone, acetyl-Orn, acetyl-diaminobutanoic acid, and acetylLys.

The invention also provides a peptide comprising a sequence of any oneof SEQ ID NOs: 100-282; SEQ ID NOs: 333-515. Also provided is a peptidecomprising a sequence of any one of SEQ ID NOs: 100-282; SEQ ID NOs:333-515 with up to 3 (e.g., 1, 2, 3) amino acid modifications, whereinthe peptide exhibits agonist activity at the human GIP receptor and thehuman GLP-1 receptor. In exemplary aspects of the peptides comprisingthe sequence of any one of SEQ ID NOs: 100-282 and SEQ ID NOs: 333-515with up to 3 (e.g., 1, 2, 3) amino acid modifications, the amino acid atposition 29 is not Gly when the amino acid at position 28 is Asp. Infurther exemplary aspects of the peptides comprising the sequence of anyone of SEQ ID NOs: 100-282 and SEQ ID NOs: 333-515 with up to 3 (e.g.,1, 2, 3) amino acid modifications, the amino acid at position 1 is anamino acid comprising a side chain imidazole, the amino acid at position2 is a DPP-IV protective amino acid, optionally, an alpha,alpha-disubstituted amino acid, the C-terminal amino acid of the peptideis amidated, and the peptide comprises at position 40 an amino acidcovalently attached to a C12 to C18 acyl or alkyl group, when the aminoacid at position 10 is Tyr, and optionally, the peptide comprises Gly atposition 41. In exemplary aspects of the peptides comprising thesequence of any one of SEQ ID NOs: 100-282 and SEQ ID NOs: 333-515 withup to 3 (e.g., 1, 2, 3) amino acid modifications, (i) the amino acid atposition 1 is His; (ii) the amino acid at position 2 is AIB; (iii) theamino acid at position 16 is Glu; (iv) the amino acid at position 17 isArg; (v) the amino acid at position 18 is Ala; (vi) the amino acid atposition 19 is Ala; (vii) the amino acid at position 20 is Gln; (viii)the amino acid at position 27 is Leu; (ix) the amino acid at position 29is Gly; (x) the amino acid at position 20 is Gly; (xi) the amino acid atposition 35 is Ala; or (xii) a combination thereof. In exemplary aspectsof the peptides comprising the sequence of any one of SEQ ID NOs:100-282 and SEQ ID NOs: 333-515 with up to 3 (e.g., 1, 2, 3) amino acidmodifications, the amino acid at position 3 is selected from the groupconsisting of: Val, norvaline, homoserine, Met, methionine sulfoxide,methionine sulfone, acetyl-Orn, acetyl-diaminobutanoic acid, and acetylLys.

In some embodiments, the glucagon analog comprises a structure based ona parent sequence comprising any one of SEQ ID NOs: 15-42, 43, 44, 52,53, 63-65, 100-312, 320, 321, 330-543, 544, 545, 553, 554, and 563-565,but differs from the parent sequence at one or more positions.

In some or any embodiments, the peptide of the present disclosures is ananalog of a parent sequence comprising any of SEQ ID NOs: 15-42, 43, 44,52, 53, 63-65, 100-312, 320, 321, 330-543, 544, 545, 553, 554, and563-565 comprising an amino acid sequence based on the amino acidsequence of the parent sequence but differs from the parent sequenceinasmuch as the amino acid sequence of the analog comprises one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and in someinstances, 16 or more (e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.),specified or optional amino acid modifications. In some or anyembodiments, the peptide of the present disclosures comprises a total of1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to9, or up to 10 additional amino acid modifications relative to theparent sequence comprising any of SEQ ID NOs: 15-42, 43, 44, 52, 53,63-65, 100-312, 320, 321, 330-543, 544, 545, 553, 554, and 563-565. Insome or any embodiments, the modifications are any of those describedherein with regard to glucagon analogs, e.g., acylation, alkylation,pegylation, truncation at C-terminus, substitution of the amino acid atone or more of positions 1, 2, 3, 7, 10, 12, 15, 16, 17, 18, 19, 20, 21,23, 24, 27, 28, and 29. For example, the peptide is an analog of aparent sequence of any one of SEQ ID NOs: 15-42, 43, 44, 52, 53, 63-65,100-312, 320, 321, 330-543, 544, 545, 553, 554, and 563-565 comprisingthe amino acid sequence of the parent sequence with up to 4, up to 5, upto 6, up to 7, up to 8, up to 9, or up to 10 additional amino acidmodifications relative to the parent sequence. In exemplary embodimentsof the analog, the amino acid at position 29 is not Gly when the aminoacid at position 28 is Asp. In further exemplary aspects of the analogs,the amino acid at position 1 is an amino acid comprising a side chainimidazole, the amino acid at position 2 is a DPP-IV protective aminoacid, optionally, an alpha, alpha-disubstituted amino acid, theC-terminal amino acid of the peptide is amidated, and the peptidecomprises at position 40 an amino acid covalently attached to a C12 toC18 acyl or alkyl group, when the amino acid at position 10 is Tyr, andoptionally, the peptide comprises Gly at position 41. In exemplaryaspects of the analogs, (i) the amino acid at position 1 is His; (ii)the amino acid at position 2 is AIB; (iii) the amino acid at position 16is Glu; (iv) the amino acid at position 17 is Arg; (v) the amino acid atposition 18 is Ala; (vi) the amino acid at position 19 is Ala; (vii) theamino acid at position 20 is Gln; (viii) the amino acid at position 27is Leu; (ix) the amino acid at position 29 is Gly; (x) the amino acid atposition 20 is Gly; (xi) the amino acid at position 35 is Ala; or (xii)a combination thereof. In exemplary aspects of the analogs, the aminoacid at position 3 is selected from the group consisting of: Val,norvaline, homoserine, Met, methionine sulfoxide, methionine sulfone,acetyl-Orn, acetyl-diaminobutanoic acid, and acetyl Lys.

In some or any embodiments, the modification is an amino acidsubstitution or replacement, e.g., a conservative amino acidsubstitution. In some aspects, the conservative substitution is areplacement of the amino acid at one or more of positions 2, 5, 7, 10,11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29. In alternativeembodiments, the amino acid substitution is not a conservative aminoacid substitution, e.g., is a non-conservative amino acid substitution.

In exemplary aspects of the analogs of a parent sequence comprising anyof SEQ ID NOs: 15-42, 43, 44, 52, 53, 63-65, 100-312, 320, 321, 330-543,544, 545, 553, 554, and 563-565 comprising an amino acid sequence basedon the amino acid sequence of the parent sequence but differs from theparent sequence, X₂₉ is not Gly when X₂₈ is Asp. In further exemplaryaspects of such analogs, X₁ is an amino acid comprising a side chainimidazole, X₂ is a DPP-IV protective amino acid, optionally, an alpha,alpha-disubstituted amino acid, the C-terminal amino acid of the peptideis amidated, and the peptide comprises at position 40 an amino acidcovalently attached to a C12 to C18 acyl or alkyl group, when X₁₀ isTyr, and optionally, the peptide comprises Gly at position 41. Inexemplary aspects of the analogs of a parent sequence comprising any ofSEQ ID NOs: 15-42, 43, 44, 52, 53, 63-65, 100-312, 320, 321, 330-543,544, 545, 553, 554, and 563-565 comprising an amino acid sequence basedon the amino acid sequence of the parent sequence but differs from theparent sequence, (i) X₁ is His; (ii) X₂ is AIB; (iii) X₁₆ is Glu; (iv)X₁₇ is Arg; (v) X₁₈ is Ala; (vi) X₁₉ is Ala; (vii) X₂₀ is Gln; (viii)X₂₇ is Leu; (ix) X₂₉ is Gly; (x) X₃₀ is Gly; (xi) X₃₅ is Ala; or (xii) acombination thereof. In exemplary aspects of the analogs of a parentsequence comprising any of SEQ ID NOs: 15-42, 43, 44, 52, 53, 63-65,100-312, 320, 321, 330-543, 544, 545, 553, 554, and 563-565 comprisingan amino acid sequence based on the amino acid sequence of the parentsequence but differs from the parent sequence, In some embodiments, thepeptide of the present disclosures comprises an amino acid sequencewhich has at least 25% sequence identity to the amino acid sequence ofthe parent sequence, which comprises any of SEQ ID NOs: 15-42, 43, 44,52, 53, 63-65, 100-312, 320, 321, 330-543, 544, 545, 553, 554, and563-565. In some embodiments, the peptide of the present disclosurescomprises an amino acid sequence which is at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90% or has greater than 90% sequence identity to the parentsequence. In some embodiments, the amino acid sequence of the presentlydisclosed peptide which has the above-referenced % sequence identity isthe full-length amino acid sequence of the presently disclosed peptide.In some embodiments, the amino acid sequence of the peptide of thepresent disclosures which has the above-referenced % sequence identityis only a portion of the amino acid sequence of the presently disclosedpeptide. In some embodiments, the presently disclosed peptide comprisesan amino acid sequence which has about A % or greater sequence identityto a reference amino acid sequence of at least 5 contiguous amino acids(e.g., at least 6, at least 7, at least 8, at least 9, at least 10 aminoacids) of the parent sequence, wherein the reference amino acid sequencebegins with the amino acid at position C of SEQ ID NO: 1 and ends withthe amino acid at position D of SEQ ID NO: 1, wherein A is 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,98, 99; C is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 and D is 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28 or 29. Any and all possible combinations of the foregoing parametersare envisioned, including but not limited to, e.g., wherein A is 90% andC and D are 1 and 27, or 6 and 27, or 8 and 27, or 10 and 27, or 12 and27, or 16 and 27.

The analogs of the parent sequence comprising any of SEQ ID NOs: 15-42,43, 44, 52, 53, 63-65, 100-312, 320, 321, 330-543, 544, 545, 553, 554,and 563-565 described herein may comprise a peptide backbone of anynumber of amino acids, i.e., can be of any peptide length. In someembodiments, the peptides described herein are the same length as SEQ IDNO: 1, i.e., are 29 amino acids in length. In some embodiments, thepresently disclosed peptide is longer than 29 amino acids in length,e.g., the presently disclosed peptide comprises a C-terminal extensionof 1-21 amino acids, as further described herein. Accordingly, thepeptide of the present disclosures in some embodiments, is 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 amino acids in length. In some embodiments, the presently disclosedpeptide is up to 50 amino acids in length. In some embodiments, thepresently disclosed peptide is longer than 29 amino acids in length(e.g., greater than 50 amino acids, (e.g., at least or about 60, atleast or about 70, at least or about 80, at least or about 90, at leastor about 100, at least or about 150, at least or about 200, at least orabout 250, at least or about 300, at least or about 350, at least orabout 400, at least or about 450, at least or about 500 amino acids inlength) due to fusion with another peptide. In other embodiments, thepresently disclosed peptide is less than 29 amino acids in length, e.g.,28, 27, 26, 25, 24, 23, amino acids.

In accordance with the foregoing, in some aspects, the peptide of thepresent disclosures is an analog of a parent sequence comprising any ofSEQ ID NOs: 15-42, 43, 44, 52, 53, 63-65, 100-312, 320, 321, 330-543,544, 545, 553, 554, and 563-565 which sequence of the analog comprisesone or more amino acid modifications which affect GIP activity, glucagonactivity, and/or GLP-1 activity, enhance stability, e.g., by reducingdegradation of the peptide (e.g., by improving resistance to DPP-IVproteases), enhance solubility, increase half-life, delay the onset ofaction, extend the duration of action at the GIP, glucagon, or GLP-1receptor, or a combination of any of the foregoing. Such amino acidmodifications, in addition to other modifications, are further describedherein with regard to glucagon analogs, and any of these modificationscan be applied individually or in combination.

Methods of Making Peptides

The glucagon analogs of the disclosure can be obtained by methods knownin the art. Suitable methods of de novo synthesizing peptides aredescribed in, for example, Chan et al., Fmoc Solid Phase PeptideSynthesis, Oxford University Press, Oxford, United Kingdom, 2005;Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc.,2000; Epitope Mapping, ed. Westwood et al., Oxford University Press,Oxford, United Kingdom, 2000; and U.S. Pat. No. 5,449,752. Additionalexemplary methods of making the peptides of the present disclosures areset forth in Example 1.

In some embodiments, the peptides described herein are commerciallysynthesized by companies, such as Synpep (Dublin, Calif.), PeptideTechnologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems(San Diego, Calif.). In this respect, the peptides can be synthetic,recombinant, isolated, and/or purified.

Also, in the instances in which the analogs of the disclosure do notcomprise any non-coded or non-natural amino acids, the glucagon analogcan be recombinantly produced using a nucleic acid encoding the aminoacid sequence of the analog using standard recombinant methods. See, forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, NY, 1994.

In some embodiments, the glucagon analogs of the disclosure areisolated. The term “isolated” as used herein means having been removedfrom its natural environment. In exemplary embodiments, the analog ismade through recombinant methods and the analog is isolated from thehost cell.

In some embodiments, the glucagon analogs of the disclosure arepurified. The term “purified,” as used herein relates to the isolationof a molecule or compound in a form that is substantially free ofcontaminants which in some aspects are normally associated with themolecule or compound in a native or natural environment and means havingbeen increased in purity as a result of being separated from othercomponents of the original composition. The purified peptide or compoundinclude, for example, peptides substantially free of nucleic acidmolecules, lipids, and carbohydrates, or other starting materials orintermediates which are used or formed during chemical synthesis of thepeptides. It is recognized that “purity” is a relative term, and not tobe necessarily construed as absolute purity or absolute enrichment orabsolute selection. In some aspects, the purity is at least or about50%, is at least or about 60%, at least or about 70%, at least or about80%, or at least or about 90% (e.g., at least or about 91%, at least orabout 92%, at least or about 93%, at least or about 94%, at least orabout 95%, at least or about 96%, at least or about 97%, at least orabout 98%, at least or about 99% or is approximately 100%.

Conjugates

The invention further provides conjugates comprising one or more of theglucagon analogs described herein conjugated to a heterologous moiety.As used herein, the term “heterologous moiety” is synonymous with theterm “conjugate moiety” and refers to any molecule (chemical orbiochemical, naturally-occurring or non-coded) which is different fromthe glucagon analogs described herein. Exemplary conjugate moieties thatcan be linked to any of the analogs described herein include but are notlimited to a heterologous peptide or polypeptide (including for example,a plasma protein), a targeting agent, an immunoglobulin or portionthereof (e.g., variable region, CDR, or Fc region), a diagnostic labelsuch as a radioisotope, fluorophore or enzymatic label, a polymerincluding water soluble polymers, or other therapeutic or diagnosticagents. In some embodiments a conjugate is provided comprising an analogof the present invention and a plasma protein, wherein the plasmaprotein is selected from the group consisting of albumin, transferin,fibrinogen and globulins. In some embodiments the plasma protein moietyof the conjugate is albumin or transferin. The conjugate in someembodiments comprises one or more of the glucagon analogs describedherein and one or more of: a peptide (which is distinct from theglucagon and/or GLP-1 receptor active glucagon analogs describedherein), a polypeptide, a nucleic acid molecule, an antibody or fragmentthereof, a polymer, a quantum dot, a small molecule, a toxin, adiagnostic agent, a carbohydrate, an amino acid.

In some embodiments, the heterologous moiety is a peptide which isdistinct from the glucagon analogs described herein and the conjugate isa fusion peptide or a chimeric peptide. In some embodiments, theheterologous moiety is a peptide extension of 1-21 amino acids. Inspecific embodiments, the extension is attached to the C-terminus of theglucagon analog, e.g., to amino acid at position 29.

In some specific aspects, the extension is a single amino acid ordipeptide. In specific embodiments, the extension comprises an aminoacid selected from the group consisting of: a charged amino acid (e.g.,a negative-charged amino acid (e.g., Glu), a positive-charged aminoacid), an amino acid comprising a hydrophilic moiety. In some aspects,the extension is Gly, Glu, Cys, Gly-Gly, Gly-Glu.

In some embodiments, the extension comprises an amino acid sequence ofSEQ ID NO: 5 (GPSSGAPPPS), SEQ ID NO: 6 (GGPSSGAPPPS), SEQ ID NO: 7(KRNRNNIA), or SEQ ID NO: 8 (KRNR). In specific aspects, the amino acidsequence is attached through the C-terminal amino acid of the glucagonanalog, e.g., amino acid at position 29. In some embodiments, the aminoacid sequence of any of SEQ ID NOs: 5-8 is bound to amino acid 29 of theglucagon analog through a peptide bond. In some specific embodiments,the amino acid at position 29 of the glucagon analog is a Gly and theGly is fused to one of the amino acid sequences of any of SEQ ID NOs:5-8.

In some embodiments, the heterologous moiety is a polymer. In someembodiments, the polymer is selected from the group consisting of:polyamides, polycarbonates, polyalkylenes and derivatives thereofincluding, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polymers of acrylic and methacrylic esters, includingpoly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate), polyvinyl polymers including polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), andpolyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, celluloses including alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt, polypropylene, polyethylenes includingpoly(ethylene glycol), poly(ethylene oxide), and poly(ethyleneterephthalate), and polystyrene.

In some aspects, the polymer is a biodegradable polymer, including asynthetic biodegradable polymer (e.g., polymers of lactic acid andglycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes,poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone)),and a natural biodegradable polymer (e.g., alginate and otherpolysaccharides including dextran and cellulose, collagen, chemicalderivatives thereof (substitutions, additions of chemical groups, forexample, alkyl, alkylene, hydroxylations, oxidations, and othermodifications routinely made by those skilled in the art), albumin andother hydrophilic proteins (e.g., zein and other prolamines andhydrophobic proteins)), as well as any copolymer or mixture thereof. Ingeneral, these materials degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion.

In some aspects, the polymer is a bioadhesive polymer, such as abioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J. A.Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In some embodiments, the polymer is a water-soluble polymer or ahydrophilic polymer. Hydrophilic polymers are further described hereinunder “Hydrophilic Moieties.” Suitable water-soluble polymers are knownin the art and include, for example, polyvinylpyrrolidone, hydroxypropylcellulose (HPC; Klucel), hydroxypropyl methylcellulose (HPMC; Methocel),nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropylbutylcellulose, hydroxypropyl pentylcellulose, methyl cellulose,ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkylcelluloses and hydroxyalkyl celluloses, various cellulose ethers,cellulose acetate, carboxymethyl cellulose, sodium carboxymethylcellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acidcopolymers, poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate,methacrylic acid copolymers, polymethacrylic acid,polymethylmethacrylate, maleic anhydride/methyl vinyl ether copolymers,poly vinyl alcohol, sodium and calcium polyacrylic acid, polyacrylicacid, acidic carboxy polymers, carboxypolymethylene, carboxyvinylpolymers, polyoxyethylene polyoxypropylene copolymer,polymethylvinylether co-maleic anhydride, carboxymethylamide, potassiummethacrylate divinylbenzene co-polymer, polyoxyethyleneglycols,polyethylene oxide, and derivatives, salts, and combinations thereof.

In specific embodiments, the polymer is a polyalkylene glycol,including, for example, polyethylene glycol (PEG).

In some embodiments, the heterologous moiety is a carbohydrate. In someembodiments, the carbohydrate is a monosaccharide (e.g., glucose,galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose),an oligosaccharide (e.g., raffinose, stachyose), or a polysaccharide(e.g., starch, amylase, amylopectin, cellulose, chitin, callose,laminarin, xylan, mannan, fucoidan, or galactomannan).

In some embodiments, the heterologous moiety is a lipid. The lipid, insome embodiments, is a fatty acid, eicosanoid, prostaglandin,leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g.,mono-, di-, tri-substituted glycerols), glycerophospholipid (e.g.,phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterollipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or apolyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin,monoglyceride, diglyceride, triglyceride, a phospholipid.

In some embodiments, the heterologous moiety is attached vianon-covalent or covalent bonding to the analog of the presentdisclosure. In exemplary aspects, the heterologous moiety is attached tothe analog of the present disclosure via a linker. Linkage can beaccomplished by covalent chemical bonds, physical forces suchelectrostatic, hydrogen, ionic, van der Waals, or hydrophobic orhydrophilic interactions. A variety of non-covalent coupling systems maybe used, including biotin-avidin, ligand/receptor, enzyme/substrate,nucleic acid/nucleic acid binding protein, lipid/lipid binding protein,cellular adhesion molecule partners; or any binding partners orfragments thereof which have affinity for each other.

The glucagon analog in some embodiments is linked to conjugate moietiesvia direct covalent linkage by reacting targeted amino acid residues ofthe analog with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues ofthese targeted amino acids. Reactive groups on the analog or conjugatemoiety include, e.g., an aldehyde, amino, ester, thiol, α-haloacetyl,maleimido or hydrazino group. Derivatizing agents include, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride or other agents known in the art.Alternatively, the conjugate moieties can be linked to the analogindirectly through intermediate carriers, such as polysaccharide orpolypeptide carriers. Examples of polysaccharide carriers includeaminodextran. Examples of suitable polypeptide carriers includepolylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof,and mixed polymers of these amino acids and others, e.g., serines, toconfer desirable solubility properties on the resultant loaded carrier.

Cysteinyl residues are most commonly reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid, chloroacetamide togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,alpha-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimideor 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),deamidation of asparagine or glutamine, acetylation of the N-terminalamine, and/or amidation or esterification of the C-terminal carboxylicacid group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the analog. Sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of tyrosine, or tryptophan, or (f) theamide group of glutamine. These methods are described in WO87/05330published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev.Biochem., pp. 259-306 (1981).

In some embodiments, the glucagon analog is conjugated to a heterologousmoiety via covalent linkage between a side chain of an amino acid of theglucagon analog and the heterologous moiety. In some embodiments, theglucagon analog is conjugated to a heterologous moiety via the sidechain of an amino acid at position 16, 17, 21, 24, or 29, a positionwithin a C-terminal extension, or the C-terminal amino acid, or acombination of these positions. In some aspects, the amino acidcovalently linked to a heterologous moiety (e.g., the amino acidcomprising a heterologous moiety) is a Cys, Lys, Orn, homo-Cys, orAc-Phe, and the side chain of the amino acid is covalently bonded to aheterologous moiety.

In some embodiments, the conjugate comprises a linker that joins theglucagon analog to the heterologous moiety. In some aspects, the linkercomprises a chain of atoms from 1 to about 60, or 1 to 30 atoms orlonger, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atomslong. In some embodiments, the chain atoms are all carbon atoms. In someembodiments, the chain atoms in the backbone of the linker are selectedfrom the group consisting of C, O, N, and S. Chain atoms and linkers maybe selected according to their expected solubility (hydrophilicity) soas to provide a more soluble conjugate. In some embodiments, the linkerprovides a functional group that is subject to cleavage by an enzyme orother catalyst or hydrolytic conditions found in the target tissue ororgan or cell. In some embodiments, the length of the linker is longenough to reduce the potential for steric hindrance. If the linker is acovalent bond or a peptidyl bond and the conjugate is a polypeptide, theentire conjugate can be a fusion protein. Such peptidyl linkers may beany length. Exemplary linkers are from about 1 to 50 amino acids inlength, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids inlength. Such fusion proteins may alternatively be produced byrecombinant genetic engineering methods known to one of ordinary skillin the art.

Conjugates: Fc Fusions

As noted above, in some embodiments, the analogs are conjugated, e.g.,fused to an immunoglobulin or portion thereof (e.g., variable region,CDR, or Fc region). Known types of immunoglobulins (Ig) include IgG,IgA, IgE, IgD or IgM. The Fc region is a C-terminal region of an Igheavy chain, which is responsible for binding to Fc receptors that carryout activities such as recycling (which results in prolonged half-life),antibody dependent cell-mediated cytotoxicity (ADCC), and complementdependent cytotoxicity (CDC).

For example, according to some definitions the human IgG heavy chain Fcregion stretches from Cys226 to the C-terminus of the heavy chain. The“hinge region” generally extends from Glu216 to Pro230 of human IgG1(hinge regions of other IgG isotypes may be aligned with the IgG1sequence by aligning the cysteines involved in cysteine bonding). The Fcregion of an IgG includes two constant domains, CH2 and CH3. The CH2domain of a human IgG Fc region usually extends from amino acids 231 toamino acid 341. The CH3 domain of a human IgG Fc region usually extendsfrom amino acids 342 to 447. References made to amino acid numbering ofimmunoglobulins or immunoglobulin fragments, or regions, are all basedon Kabat et al. 1991, Sequences of Proteins of Immunological Interest,U.S. Department of Public Health, Bethesda, Md. In a relatedembodiments, the Fc region may comprise one or more native or modifiedconstant regions from an immunoglobulin heavy chain, other than CHI, forexample, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4regions of IgE.

Suitable conjugate moieties include portions of immunoglobulin sequencethat include the FcRn binding site. FcRn, a salvage receptor, isresponsible for recycling immunoglobulins and returning them tocirculation in blood. The region of the Fc portion of IgG that binds tothe FcRn receptor has been described based on X-ray crystallography(Burmeister et al. 1994, Nature 372:379). The major contact area of theFc with the FcRn is near the junction of the CH2 and CH3 domains.Fc-FcRn contacts are all within a single Ig heavy chain. The majorcontact sites include amino acid residues 248, 250-257, 272, 285, 288,290-291, 308-311, and 314 of the CH2 domain and amino acid residues385-387, 428, and 433-436 of the CH3 domain.

Some conjugate moieties may or may not include FcγR binding site(s).FcγR are responsible for ADCC and CDC. Examples of positions within theFc region that make a direct contact with FcγR are amino acids 234-239(lower hinge region), amino acids 265-269 (B/C loop), amino acids297-299 (C′/E loop), and amino acids 327-332 (F/G) loop (Sondermann etal., Nature 406: 267-273, 2000). The lower hinge region of IgE has alsobeen implicated in the FcRI binding (Henry, et al., Biochemistry 36,15568-15578, 1997). Residues involved in IgA receptor binding aredescribed in Lewis et al., (J. Immunol. 175:6694-701, 2005). Amino acidresidues involved in IgE receptor binding are described in Sayers et al.(J Biol. Chem. 279(34):35320-5, 2004).

Amino acid modifications may be made to the Fc region of animmunoglobulin. Such variant Fc regions comprise at least one amino acidmodification in the CH3 domain of the Fc region (residues 342-447)and/or at least one amino acid modification in the CH2 domain of the Fcregion (residues 231-341). Mutations believed to impart an increasedaffinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al.2001, J. Biol. Chem. 276:6591). Other mutations may reduce binding ofthe Fc region to FcγRI, FcγRIIA, FcγRIIB, and/or FcγRIIIA withoutsignificantly reducing affinity for FcRn. For example, substitution ofthe Asn at position 297 of the Fc region with Ala or another amino acidremoves a highly conserved N-glycosylation site and may result inreduced immunogenicity with concomitant prolonged half-life of the Fcregion, as well as reduced binding to FcγRs (Routledge et al. 1995,Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632;Shields et al. 1995, J. Biol. Chem. 276:6591). Amino acid modificationsat positions 233-236 of IgG1 have been made that reduce binding to FcγR5(Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613). Some exemplary amino acid substitutionsare described in U.S. Pat. Nos. 7,355,008 and 7,381,408, eachincorporated by reference herein in its entirety.

Conjugates: Hydrophilic Moieties

The glucagon analogs described herein can be further modified to improveits solubility and stability in aqueous solutions at physiological pH,while retaining the high biological activity relative to nativeglucagon. Hydrophilic moieties such as PEG groups can be attached to theanalogs under any suitable conditions used to react a protein with anactivated polymer molecule. Any means known in the art can be used,including via acylation, reductive alkylation, Michael addition, thiolalkylation or other chemoselective conjugation/ligation methods througha reactive group on the PEG moiety (e.g., an aldehyde, amino, ester,thiol, α-haloacetyl, maleimido or hydrazino group) to a reactive groupon the target compound (e.g., an aldehyde, amino, ester, thiol,α-haloacetyl, maleimido or hydrazino group). Activating groups which canbe used to link the water soluble polymer to one or more proteinsinclude without limitation sulfone, maleimide, sulfhydryl, thiol,triflate, tresylate, azidirine, oxirane, 5-pyridyl, andalpha-halogenated acyl group (e.g., alpha-iodo acetic acid,alpha-bromoacetic acid, alpha-chloroacetic acid). If attached to theanalog by reductive alkylation, the polymer selected should have asingle reactive aldehyde so that the degree of polymerization iscontrolled. See, for example, Kinstler et al., Adv. Drug. Delivery Rev.54: 477-485 (2002); Roberts et al., Adv. Drug Delivery Rev. 54: 459-476(2002); and Zalipsky et al., Adv. Drug Delivery Rev. 16: 157-182 (1995).

In specific aspects, an amino acid residue of the analog having a thiolis modified with a hydrophilic moiety such as PEG. In some embodiments,the thiol is modified with maleimide-activated PEG in a Michael additionreaction to result in a PEGylated analog comprising the thioetherlinkage shown below:

In some embodiments, the thiol is modified with a haloacetyl-activatedPEG in a nucleophilic substitution reaction to result in a PEGylatedanalog comprising a thioether linkage.

Suitable hydrophilic moieties include polyethylene glycol (PEG),polypropylene glycol, polyoxyethylated polyols (e.g., POG),polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylatedglycerol (POG), polyoxyalkylenes, polyethylene glycol propionaldehyde,copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethyleneglycol, mono-(C1-C10)alkoxy- or aryloxy-polyethylene glycol,carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, poly(β-amino acids) (either homopolymers or randomcopolymers), poly(n-vinyl pyrrolidone)polyethylene glycol, propropyleneglycol homopolymers (PPG) and other polyakylene oxides, polypropyleneoxide/ethylene oxide copolymers, colonic acids or other polysaccharidepolymers, Ficoll or dextran and mixtures thereof. Dextrans arepolysaccharide polymers of glucose subunits, predominantly linked byα1-6 linkages. Dextran is available in many molecular weight ranges,e.g., about 1 kD to about 100 kD, or from about 5, 10, 15 or 20 kD toabout 20, 30, 40, 50, 60, 70, 80 or 90 kD. Linear or branched polymersare contemplated. Resulting preparations of conjugates may beessentially monodisperse or polydisperse, and may have about 0.5, 0.7,1, 1.2, 1.5 or 2 polymer moieties per analog.

In some or any embodiments, the glucagon analog is conjugated to ahydrophilic moiety via covalent linkage between a side chain of an aminoacid of the glucagon analog and the hydrophilic moiety. In some or anyembodiments, the glucagon analog is conjugated to a hydrophilic moietyvia the side chain of an amino acid at position 16, 17, 21, 24, or 29, aposition within a C-terminal extension, or the C-terminal amino acid, ora combination of these positions. In some aspects, the amino acidcovalently linked to a hydrophilic moiety (e.g., the amino acidcomprising a hydrophilic moiety) is a Cys, Lys, Orn, homo-Cys, orAc-Phe, and the side chain of the amino acid is covalently bonded to ahydrophilic moiety (e.g., PEG).

Conjugates: rPEG

In some or any embodiments, the conjugate of the present disclosurecomprises the glucagon analog having GIP receptor agonist activity fusedto an accessory analog which is capable of forming an extendedconformation similar to chemical PEG (e.g., a recombinant PEG (rPEG)molecule), such as those described in International Patent ApplicationPublication No. WO2009/023270 and U.S. Patent Application PublicationNo. US20080286808. The rPEG molecule in some aspects is a polypeptidecomprising one or more of glycine, serine, glutamic acid, aspartic acid,alanine, or proline. In some aspects, the rPEG is a homopolymer, e.g.,poly-glycine, poly-serine, poly-glutamic acid, poly-aspartic acid,poly-alanine, or poly-proline. In other embodiments, the rPEG comprisestwo types of amino acids repeated, e.g., poly(Gly-Ser), poly(Gly-Glu),poly(Gly-Ala), poly(Gly-Asp), poly(Gly-Pro), poly(Ser-Glu), etc. In someaspects, the rPEG comprises three different types of amino acids, e.g.,poly(Gly-Ser-Glu). In specific aspects, the rPEG increases the half-lifeof the Glucagon and/or GLP-1 agonist analog. In some aspects, the rPEGcomprises a net positive or net negative charge. The rPEG in someaspects lacks secondary structure. In some embodiments, the rPEG isgreater than or equal to 10 amino acids in length and in someembodiments is about 40 to about 50 amino acids in length. The accessorypeptide in some aspects is fused to the N- or C-terminus of the analogof the present disclosure through a peptide bond or a proteinasecleavage site, or is inserted into the loops of the analog of thepresent disclosure. The rPEG in some aspects comprises an affinity tagor is linked to a PEG that is greater than 5 kDa. In some embodiments,the rPEG confers the analog of the present disclosure with an increasedhydrodynamic radius, serum half-life, protease resistance, or solubilityand in some aspects confers the analog with decreased immunogenicity.

Conjugates: Multimers

The invention further provides multimers or dimers of the analogsdisclosed herein, including homo- or hetero-multimers or homo- orhetero-dimers. Two or more of the analogs can be linked together usingstandard linking agents and procedures known to those skilled in theart. For example, dimers can be formed between two peptides through theuse of bifunctional thiol crosslinkers and bi-functional aminecrosslinkers, particularly for the analogs that have been substitutedwith cysteine, lysine ornithine, homocysteine or acetyl phenylalanineresidues. The dimer can be a homodimer or alternatively can be aheterodimer. In exemplary embodiments, the linker connecting the two (ormore) analogs is PEG, e.g., a 5 kDa PEG, 20 kDa PEG. In someembodiments, the linker is a disulfide bond. For example, each monomerof the dimer may comprise a Cys residue (e.g., a terminal or internallypositioned Cys) and the sulfur atom of each Cys residue participates inthe formation of the disulfide bond. In exemplary aspects, each monomerof the dimer is linked via a thioether bond. In exemplary aspects, anepsilon amine of a Lys residue of one monomer is bonded to a Cysresidue, which, in turn, is connected via a chemical moiety to theepsilon amine of a Lys residue of the other monomer. Methods of makingsuch thioether bonded dimers are further described herein. In someaspects, the monomers are connected via terminal amino acids (e.g.,N-terminal or C-terminal), via internal amino acids, or via a terminalamino acid of at least one monomer and an internal amino acid of atleast one other monomer. In specific aspects, the monomers are notconnected via an N-terminal amino acid. In some aspects, the monomers ofthe multimer are attached together in a “tail-to-tail” orientation inwhich the C-terminal amino acids of each monomer are attached together.

Prodrugs

Further provided by the invention are prodrugs of the peptides andanalogs described herein. As used herein, the term “prodrug” is definedas any compound that undergoes chemical modification before exhibitingits full pharmacological effects.

In exemplary embodiments, the prodrug is an amide-based peptide prodrug,similar to those described in International Patent ApplicationPublication No. WO/2010/071807, which published on Jun. 24, 2010. Suchprodrugs are intended to delay onset of action and extend the half lifeof the drug. The delayed onset of action is advantageous in that itallows systemic distribution of the prodrug prior to its activation.Accordingly, the administration of prodrugs eliminates complicationscaused by peak activities upon administration and increases thetherapeutic index of the parent drug.

In exemplary aspects, the prodrug comprises the structure: A-B-Q;wherein Q is a peptide or analog described herein; A is an amino acid ora hydroxy acid; B is an N-alkylated amino acid linked to Q through anamide bond between A-B and an amine of Q; wherein A, B, or the aminoacid of Q to which A-B is linked is a non-coded amino acid, furtherwherein chemical cleavage half-life (t1/2) of A-B from Q is at leastabout 1 hour to about 1 week in PBS under physiological conditions. Asused herein the term “hydroxy acid” refers to an amino acid that hasbeen modified to replace the alpha carbon amino group with a hydroxylgroup.

In some embodiments the dipeptide prodrug element has the generalstructure of Formula I:

wherein

R₁, R₂, R₄ and R₈ are independently selected from the group consistingof H, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)SH,(C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl),(C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(A)(W₁)C₁-C₁₂ alkyl, whereinW₁ is a heteroatom selected from the group consisting of N, S and O, orR₁ and R₂ together with the atoms to which they are attached form aC3-C12 cycloalkyl or aryl; or R₄ and R₈ together with the atoms to whichthey are attached form a C3-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₁₈ alkyl, (C₁-C₁₈alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉ heteroaryl) or R₄ and R₃together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl or R₆ and R₂ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of H and OH.

In other embodiments the dipeptide prodrug element has the generalstructure of Formula I:

wherein

R₁, R₂, R₄ and R₈ are independently selected from the group consistingof H, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)SH,(C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl),(C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(W₁)C₁-C₁₂ alkyl, wherein W₁is a heteroatom selected from the group consisting of N, S and O, or R₁and R₂ together with the atoms to which they are attached form a C₃-C₁₂cycloalkyl; or R₄ and R₈ together with the atoms to which they areattached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₁₈ alkyl, (C₁-C₁₈alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉ heteroaryl) or R₄ and R₃together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl or R₆ and R₁ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

In some embodiments R₈ is H and R₅ is NHR₆.

In some embodiments the dipeptide prodrug element has the structure ofFormula I, wherein

R₁ and R₈ are independently H or C₁-C₈ alkyl;

R₂ and R₄ are independently selected from the group consisting of H,C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₂-C₃alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄ alkyl)NH₂,(C₁-C₄ alkyl)NHC(NH₂+) NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and CH₂(C₃-C₉heteroaryl), or R₁ and R₂ together with the atoms to which they areattached form a C₃-C₁₂ cycloalkyl or aryl;

R₅ is NHR₆; and

R₆ is H or C₁-C₈ alkyl.

In other embodiments the dipeptide prodrug element has the structure ofFormula I,

wherein

R₁ and R₈ are independently H or C₁-C₈ alkyl;

R₂ and R₄ are independently selected from the group consisting of H,C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₂-C₃alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄ alkyl)NH₂,(C₁-C₄ alkyl)NHC(NH₂+) NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and CH₂(C₃-C₉heteroaryl), or R₁ and R₂ together with the atoms to which they areattached form a C₃-C₁₂ cycloalkyl;

R₃ is C₁-C₁₈ alkyl;

R₅ is NHR₆;

R₆ is H or C₁-C₈ alkyl; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

The half life of the prodrug formed in accordance with the presentdisclosure is determined by the substituents of the dipeptide prodrugelement, its location, and the amino acid to which it is attached. Forexample, the prodrug may comprise a peptide or analog described herein,wherein the dipeptide prodrug element is linked through the alpha aminogroup of the N-terminal amino acid of the peptide or analog describedherein. In this embodiment prodrugs having a t_(1/2) of, e.g., about 1hour comprise a dipeptide prodrug element with the structure:

wherein

R₁ and R₂ are independently C₁-C₁₈ alkyl or aryl; or R₁ and R₂ arelinked through —(CH₂)_(p), wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen; and

R₅ is an amine.

In other embodiments, prodrugs having a t_(1/2) of, e.g., about 1 hourcomprise a dipeptide prodrug element with the structure:

wherein

R₁ and R₂ are independently C₁-C₁₈ alkyl or (C₀-C₄ alkyl)(C₆-C₁₀aryl)R₇; or R₁ and R₂ are linked through —(CH₂)_(p), wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen;

R₅ is NH₂; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

Furthermore, prodrugs having the dipeptide prodrug element linked to theN-terminal alpha amino acid of the peptide or analog described hereinand having a t_(1/2), e.g., between about 6 to about 24 hours, comprisea dipeptide prodrug element with the structure:

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, C₁-C₁₈ alkyl and aryl, or R₁ and R₂ are linked through(CH₂)_(p), wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-12 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₈ alkyl and aryl; and R₅ is an amine; with the provisothat both R₁ and R₂ are not hydrogen and provided that one of R₄ or R₈is hydrogen.

In some embodiments, prodrugs having the dipeptide prodrug elementlinked to the N-terminal alpha amino acid of the peptide or analogdescribed herein and having a t_(1/2), e.g., between about 12 to about72 hours, or in some embodiments between about 12 to about 48 hours,comprise a dipeptide prodrug element with the structure:

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₄ alkyl)NH₂, and(C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, or R₁ and R₂ are linked through (CH₂)_(p),wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-12 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₈ alkyl and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₅ is NH₂; and

R₇ is selected from the group consisting of H, C₁-C₁₈ alkyl, C₂-C₁₈alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂, (C₀-C₄alkyl)OH, and halo;

with the proviso that both R₁ and R₂ are not hydrogen and provided thatat least one of R₄ or R₈ is hydrogen.

In some embodiments, prodrugs having the dipeptide prodrug elementlinked to the N-terminal amino acid of the peptide or analog describedherein and having a t_(1/2), e.g., between about 12 to about 72 hours,or in some embodiments between about 12 to about 48 hours, comprise adipeptide prodrug element with the structure:

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, C₁-C₈ alkyl and (C₁-C₄ alkyl)NH₂, or R₁ and R₂ are linkedthrough (CH₂)_(p), wherein p is 2-9;

R₃ is C₁-C₈ alkyl or R₃ and R₄ together with the atoms to which they areattached form a 4-6 heterocyclic ring;

R₄ is selected from the group consisting of hydrogen and C₁-C₈ alkyl;and

R₅ is NH₂;

with the proviso that both R₁ and R₂ are not hydrogen.

In other embodiments, prodrugs having the dipeptide prodrug elementlinked to the N-terminal amino acid of the peptide or analog describedhere and having a t_(1/2), e.g., between about 12 to about 72 hours, orin some embodiments between about 12 to about 48 hours, comprise adipeptide prodrug element with the structure:

wherein

-   -   R₁ and R₂ are independently selected from the group consisting        of hydrogen, C₁-C₈ alkyl and (C₁-C₄ alkyl)NH₂;    -   R₃ is C₁-C₆ alkyl;    -   R₄ is hydrogen; and    -   R₅ is NH₂;    -   with the proviso that both R₁ and R₂ are not hydrogen.

In some embodiments, prodrugs having the dipeptide prodrug elementlinked to the N-terminal amino acid of the peptide or analog describedherein and having a t_(1/2), e.g., between about 12 to about 72 hours,or in some embodiments between about 12 to about 48 hours, comprise adipeptide prodrug element with the structure:

wherein

-   -   R₁ and R₂ are independently selected from the group consisting        of hydrogen and C₁-C₈ alkyl, (C₁-C₄ alkyl)NH₂, or R₁ and R₂ are        linked through (CH₂)_(p), wherein p is 2-9;    -   R₃ is C₁-C₈ alkyl;    -   R₄ is (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;    -   R₅ is NH₂; and    -   R₇ is selected from the group consisting of hydrogen, C₁-C₈        alkyl and (C₀-C₄ alkyl)OH;    -   with the proviso that both R₁ and R₂ are not hydrogen.

In addition a prodrug having the dipeptide prodrug element linked to theN-terminal alpha amino acid of the peptide and analog described hereinand having a t_(1/2), e.g., of about 72 to about 168 hours is providedwherein the dipeptide prodrug element has the structure:

wherein R₁ is selected from the group consisting of hydrogen, C₁-C₈alkyl and aryl;

-   -   R₃ is C₁-C₁₈ alkyl;    -   R₄ and R₈ are each hydrogen; and    -   R₅ is an amine or N-substituted amine or a hydroxyl;        with the proviso that, if R₁ is alkyl or aryl, then R₁ and R₅        together with the atoms to which they are attached form a 4-11        heterocyclic ring.

In some embodiments, the dipeptide prodrug element has the structure:

wherein R₁ is selected from the group consisting of hydrogen, C₁-C₈alkyl and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl, or R₆ and R₁ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo;

with the proviso that, if R₁ is alkyl or (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇,then R₁ and R₅ together with the atoms to which they are attached form a4-11 heterocyclic ring.

In some embodiments the dipeptide prodrug element is linked to a sidechain amine of an internal amino acid of the peptide or analog describedherein. In this embodiment prodrugs having a t_(1/2), e.g., of about 1hour have the structure:

wherein

-   -   R₁ and R₂ are independently C₁-C₈ alkyl or aryl; or R₁ and R₂        are linked through (CH₂)_(p), wherein p is 2-9;    -   R₃ is C₁-C₁₈ alkyl;    -   R₄ and R₈ are each hydrogen; and R₅ is an amine.

In some embodiments, prodrugs having a t_(1/2), e.g., of about 1 hourhave the structure:

wherein

-   -   R₁ and R₂ are independently C₁-C₈ alkyl or (C₀-C₄ alkyl)(C₆-C₁₀        aryl)R₇; or R₁ and R₂ are linked through —(CH₂)_(p)—, wherein p        is 2-9;    -   R₃ is C₁-C₁₈ alkyl;    -   R₄ and R₈ are each hydrogen;    -   R₅ is NH₂; and    -   R₇ is selected from the group consisting of hydrogen, C₁-C₁₈        alkyl, C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH,        (C₀-C₄ alkyl)NH₂, (C₀-C₄ alkyl)OH, and halo.

Furthermore, prodrugs having a t_(1/2), e.g., between about 6 to about24 hours and having the dipeptide prodrug element linked to a internalamino acid side chain comprise a dipeptide prodrug element with thestructure:

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, C₁-C₈ alkyl and aryl, or R₁ and R₂ are linked through—(CH₂)_(p), wherein p is 2-9;

-   -   R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which        they are attached form a 4-12 heterocyclic ring;    -   R₄ and R₈ are independently C₁-C₁₈ alkyl or aryl; and    -   R₅ is an amine or N-substituted amine;    -   with the proviso that both R₁ and R₂ are not hydrogen and        provided that one of R₄ or R₈ is hydrogen.

In some embodiments, prodrugs having a t_(1/2), e.g., between about 12to about 72 hours, or in some embodiments between about 12 to about 48hours, and having the dipeptide prodrug element linked to a internalamino acid side chain comprise a dipeptide prodrug element with thestructure:

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, C₁-C₈ alkyl, and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, or R₁ and R₂are linked through —(CH₂)_(p)—, wherein p is 2-9;

-   -   R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which        they are attached form a 4-12 heterocyclic ring;    -   R₄ and R₈ are independently hydrogen, C₁-C₁₈ alkyl or (C₀-C₄        alkyl)(C₆-C₁₀ aryl)R₇;    -   R₅ is NHR₆;    -   R₆ is H or C₁-C₈ alkyl, or R₆ and R₂ together with the atoms to        which they are attached form a 4, 5 or 6 member heterocyclic        ring; and    -   R₇ is selected from the group consisting of hydrogen, C₁-C₁₈        alkyl, C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH,        (C₀-C₄ alkyl)NH₂, (C₀-C₄ alkyl)OH, and halo;    -   with the proviso that both R₁ and R₂ are not hydrogen and        provided that at least one of R₄ or R₈ is hydrogen.

In addition a prodrug having a t_(1/2), e.g., of about 72 to about 168hours and having the dipeptide prodrug element linked to a internalamino acid side chain of the peptide or analog described herein isprovided wherein the dipeptide prodrug element has the structure:

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, C₁-C₁₈ alkyl and aryl;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen; and

R₅ is an amine or N-substituted amine or a hydroxyl;

with the proviso that, if R₁ and R₂ are both independently an alkyl oraryl, either R₁ or R₂ is linked through (CH2)_(p) to R₅, wherein p is2-9.

In some embodiments, a prodrug having a t_(1/2), e.g., of about 72 toabout 168 hours and having the dipeptide prodrug element linked to ainternal amino acid side chain of the peptide or analog described hereinis provided wherein the dipeptide prodrug element has the structure:

wherein R₁ is selected from the group consisting of hydrogen, C₁-C₁₈alkyl and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen;

R₅ is NHR₆ or OH;

R₆ is H or C₁-C₈ alkyl, or R₆ and R₁ together with the atoms to whichthey are attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo;with the proviso that, if R₁ and R₂ are both independently an alkyl or(C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, either R₁ or R₂ is linked through(CH₂)_(p) to R₅, wherein p is 2-9.

In some embodiments the dipeptide prodrug element is linked to a sidechain amine of an internal amino acid of the peptide or analog describedherein wherein the internal amino acid comprises the structure ofFormula II:

wherein

n is an integer selected from 1 to 4. In some embodiments n is 3 or 4and in some embodiments the internal amino acid is lysine. In someembodiments the dipeptide prodrug element is linked to a primary amineon a side chain of an amino acid located at position 12, 16, 17, 18, 20,28, or 29 of the peptide or analog described herein. In some embodimentsthe amino acid at 12, 16, 17, 18, 20, 28, or 29 comprises the structureof Formula II:

wherein n is an integer selected from 1 to 4 and the dipeptide prodrugelement is linked to the amino acid side chain via an amide bond. Insome embodiments n is 4 and the amino acid is located at position 20.

In a further embodiment the dipeptide prodrug element is linked to thepeptide or analog thereof via an amine present on an aryl group of anaromatic amino acid. In some embodiments the aromatic amino acid is aninternal amino acid of the peptide or analog described herein, howeverthe aromatic amino acid can also be the N-terminal amino acid. In someembodiments the aromatic amino acid is selected from the groupconsisting of amino-Phe, amino-napthyl alanine, amino tryptophan,amino-phenyl-glycine, amino-homo-Phe, and amino tyrosine. In someembodiments the primary amine that forms an amide bond with thedipeptide prodrug element is in the para-position on the aryl group. Insome embodiments the aromatic amine comprises the structure of FormulaIII:

-   -   wherein m is an integer from 1 to 3.

For those embodiments wherein the dipeptide prodrug element is linked tothe peptide or analog described herein via an amine present on an arylgroup of an aromatic amino acid, prodrugs having a t_(1/2), e.g., ofabout 1 hour have a dipeptide structure of:

wherein R₁ and R₂ are independently C₁-C₁₈ alkyl or aryl;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-12 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₁₈ alkyl and aryl; and R₅ is an amine or a hydroxyl.

In some embodiments, the dipeptide prodrug element is linked to thepeptide or analog described herein via an amine present on an aryl groupof an aromatic amino acid, prodrugs having a t_(1/2), e.g., of about 1hour have a dipeptide structure of:

wherein R₁ and R₂ are independently C₁-C₁₈ alkyl or (C₀-C₄ alkyl)(C₆-C₁₀aryl)R₇;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-12 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₁₈ alkyl and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₅ is NH₂ or OH; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

Furthermore, prodrugs having the dipeptide prodrug element is linked viaan aromatic amino acid and having a t_(1/2), e.g., of about 6 to about24 hours are provided wherein the dipeptide comprises a structure of:

wherein

R₁ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl andaryl, or R₁ and R₂ are linked through —(CH₂)_(p), wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-6 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₁₈ alkyl and aryl; and R₅ is an amine or N-substitutedamine.

In some embodiments, prodrugs having the dipeptide prodrug elementlinked via an aromatic amino acid and having a t_(1/2), e.g., of about 6to about 24 hours are provided wherein the dipeptide comprises astructure of:

wherein

R₁ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,(C₁-C₁₈ alkyl)OH, (C₁-C₄ alkyl)NH₂, and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-6 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₁₈ alkyl and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₅ is NHR₆;

R₆ is H, C₁-C₈ alkyl, or R₆ and R₁ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

In addition, prodrugs having the dipeptide prodrug element is linked viaan aromatic amino acid and having a t_(1/2), e.g., of about 72 to about168 hours are provided wherein the dipeptide comprises a structure of:

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, C₁-C₈ alkyl and aryl;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-6 heterocyclic ring;

R₄ and R₈ are each hydrogen; and

R₅ is selected from the group consisting of amine, N-substituted amineand hydroxyl.

In some embodiments, prodrugs having the dipeptide prodrug elementlinked via an aromatic amino acid and having a t_(1/2), e.g., of about72 to about 168 hours are provided wherein the dipeptide comprises astructure of:

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, C₁-C₈ alkyl, (C₁-C₄ alkyl)COOH, and (C₀-C₄ alkyl)(C₆-C₁₀aryl)R₇, or R₁ and R₅ together with the atoms to which they are attachedform a 4-11 heterocyclic ring;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-6 heterocyclic ring;

R₄ is hydrogen or forms a 4-6 heterocyclic ring with R₃;

R₈ is hydrogen;

R₅ is NHR₆ or OH;

R₆ is H or C₁-C₈ alkyl, or R₆ and R₁ together with the atoms to whichthey are attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

In some embodiments the dipeptide prodrug element is linked to anaromatic amino acid via a primary amine present as an aryl substituentof the aromatic amino acid, wherein the aromatic amino acid is locatedat position 10, 13, 22, or 25 of the peptide or analog described herein(based on the numbering for native glucagon, see e.g., SEQ ID NO: 1). Insome embodiments the dipeptide prodrug element linked aromatic aminoacid amino acid is located at position 22 of the peptide or analogdescribed herein.

In accordance with some embodiments the dipeptide prodrug element islinked at the N-terminal amine of the peptide or analog describedherein, including for example a glucagon related peptide, orosteocalcin, as well as analogs, derivatives and conjugates of theforegoing, wherein the dipeptide prodrug element comprises thestructure:

wherein R₁ is selected from the group consisting of H and C₁-C₈ alkyl;

R₂ and R₄ are independently selected from the group consisting of H,C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₂-C₃alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄ alkyl)NH₂,(C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, CH₂(C₅-C₉ heteroaryl), or R₁ and R₂ together withthe atoms to which they are attached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₈ alkyl,(C₃-C₆)cycloalkyl or R₄ and R₃ together with the atoms to which they areattached form a 5 or 6 member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, or R₆ and R₂ together with the atoms to which they are attachedform a 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of H and OH. In someembodiments R₁ is H or C₁-C₈ alkyl, R₂ is selected from the groupconsisting of H, C₁-C₆ alkyl, CH₂OH, (C₁-C₄ alkyl)NH₂, (C₃-C₆cycloalkyl) and CH₂(C₆ aryl)R₇ or R₆ and R₂ together with the atoms towhich they are attached form a 5 member heterocyclic ring, R₃ is C₁-C₆alkyl, and R₄ is selected from the group consisting of H, C₁-C₄ alkyl,(C₃-C₆)cycloalkyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH and (C₀-C₄ alkyl)(C₆aryl)R₇, or R₃ and R₄ together with the atoms to which they are attachedform a 5 member heterocyclic ring. In a further embodiment R₃ is CH₃, R₅is NHR₆, and in an alternative further embodiment R₃ and R₄ togetherwith the atoms to which they are attached form a 5 member heterocyclicring and R₅ is NHR₆.

In accordance with another embodiment the dipeptide prodrug element islinked at the N-terminal amine of the peptide or analog describedherein, wherein the dipeptide prodrug element comprises the structure:

wherein R₁ is selected from the group consisting of H and C₁-C₈ alkyl;

R₂ and R₄ are independently selected from the group consisting of H,C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₂-C₃alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄ alkyl)NH₂,(C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, CH₂(C₅-C₉ heteroaryl), or R₁ and R₂ together withthe atoms to which they are attached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₈ alkyl,(C₃-C₆)cycloalkyl or R₄ and R₃ together with the atoms to which they areattached form a 5 or 6 member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, or R₆ and R₂ together with the atoms to which they are attachedform a 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo. In some embodiments R₁ is H or C₁-C₈ alkyl,R₂ is selected from the group consisting of H, C₁-C₆ alkyl, CH₂OH,(C₁-C₄ alkyl)NH₂, (C₃-C₆ cycloalkyl) and CH₂(C₆ aryl)R₇ or R₆ and R₂together with the atoms to which they are attached form a 5 memberheterocyclic ring, R₃ is C₁-C₆ alkyl, and R₄ is selected from the groupconsisting of H, C₁-C₄ alkyl, (C₃-C₆)cycloalkyl, (C₁-C₄ alkyl)OH, (C₁-C₄alkyl)SH and (C₀-C₄ alkyl)(C₆ aryl)R₇, or R₃ and R₄ together with theatoms to which they are attached form a 5 member heterocyclic ring. In afurther embodiment R₃ is CH₃, R₅ is NHR₆, and in an alternative furtherembodiment R₃ and R₄ together with the atoms to which they are attachedform a 5 member heterocyclic ring and R₅ is NHR₆.

Pharmaceutical Compositions, Uses and Kits Salts

In some embodiments, the glucagon analog is in the form of a salt, e.g.,a pharmaceutically acceptable salt. As used herein the term“pharmaceutically acceptable salt” refers to salts of compounds thatretain the biological activity of the parent compound, and which are notbiologically or otherwise undesirable. Such salts can be prepared insitu during the final isolation and purification of the analog, orseparately prepared by reacting a free base function with a suitableacid. Many of the compounds disclosed herein are capable of forming acidand/or base salts by virtue of the presence of amino and/or carboxylgroups or groups similar thereto.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Representative acid addition salts include,but are not limited to acetate, adipate, alginate, citrate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate,hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate (isothionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, palmitoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate, and undecanoate. Salts derived frominorganic acids include hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like. Salts derived fromorganic acids include acetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid,maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluene-sulfonic acid, salicylic acid, and the like. Examples of acidswhich can be employed to form pharmaceutically acceptable acid additionsalts include, for example, an inorganic acid, e.g., hydrochloric acid,hydrobromic acid, sulphuric acid, and phosphoric acid, and an organicacid, e.g., oxalic acid, maleic acid, succinic acid, and citric acid.

Basic addition salts also can be prepared in situ during the finalisolation and purification of the source of salicylic acid, or byreacting a carboxylic acid-containing moiety with a suitable base suchas the hydroxide, carbonate, or bicarbonate of a pharmaceuticallyacceptable metal cation or with ammonia or an organic primary,secondary, or tertiary amine. Pharmaceutically acceptable salts include,but are not limited to, cations based on alkali metals or alkaline earthmetals such as lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like, and nontoxic quaternary ammonia and aminecations including ammonium, tetramethylammonium, tetraethylammonium,methylammonium, dimethylammonium, trimethylammonium, triethylammonium,diethylammonium, and ethylammonium, amongst others. Other representativeorganic amines useful for the formation of base addition salts include,for example, ethylenediamine, ethanolamine, diethanolamine, piperidine,piperazine, and the like. Salts derived from organic bases include, butare not limited to, salts of primary, secondary and tertiary amines.

Further, basic nitrogen-containing groups can be quaternized with theanalog of the present disclosure as lower alkyl halides such as methyl,ethyl, propyl, and butyl chlorides, bromides, and iodides; long chainhalides such as decyl, lauryl, myristyl, and stearyl chlorides,bromides, and iodides; arylalkyl halides like benzyl and phenethylbromides and others. Water or oil-soluble or dispersible products arethereby obtained.

Formulations

In accordance with some embodiments, a pharmaceutical composition isprovided wherein the composition comprises a glucagon analog of thepresent disclosure, or pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable carrier” includes any of the standardpharmaceutical carriers, such as a phosphate buffered saline solution,water, emulsions such as an oil/water or water/oil emulsion, and varioustypes of wetting agents. The term also encompasses any of the agentsapproved by a regulatory agency of the US Federal government or listedin the US Pharmacopeia for use in animals, including humans.

The pharmaceutical composition can comprise any pharmaceuticallyacceptable ingredient, including, for example, acidifying agents,additives, adsorbents, aerosol propellants, air displacement agents,alkalizing agents, anticaking agents, anticoagulants, antimicrobialpreservatives, antioxidants, antiseptics, bases, binders, bufferingagents, chelating agents, coating agents, coloring agents, desiccants,detergents, diluents, disinfectants, disintegrants, dispersing agents,dissolution enhancing agents, dyes, emollients, emulsifying agents,emulsion stabilizers, fillers, film forming agents, flavor enhancers,flavoring agents, flow enhancers, gelling agents, granulating agents,humectants, lubricants, mucoadhesives, ointment bases, ointments,oleaginous vehicles, organic bases, pastille bases, pigments,plasticizers, polishing agents, preservatives, sequestering agents, skinpenetrants, solubilizing agents, solvents, stabilizing agents,suppository bases, surface active agents, surfactants, suspendingagents, sweetening agents, therapeutic agents, thickening agents,tonicity agents, toxicity agents, viscosity-increasing agents,water-absorbing agents, water-miscible cosolvents, water softeners, orwetting agents.

In some embodiments, the pharmaceutical composition comprises any one ora combination of the following components: acacia, acesulfame potassium,acetyltributyl citrate, acetyltriethyl citrate, agar, albumin, alcohol,dehydrated alcohol, denatured alcohol, dilute alcohol, aleuritic acid,alginic acid, aliphatic polyesters, alumina, aluminum hydroxide,aluminum stearate, amylopectin, α-amylose, ascorbic acid, ascorbylpalmitate, aspartame, bacteriostatic water for injection, bentonite,bentonite magma, benzalkonium chloride, benzethonium chloride, benzoicacid, benzyl alcohol, benzyl benzoate, bronopol, butylatedhydroxyanisole, butylated hydroxytoluene, butylparaben, butylparabensodium, calcium alginate, calcium ascorbate, calcium carbonate, calciumcyclamate, dibasic anhydrous calcium phosphate, dibasic dehydratecalcium phosphate, tribasic calcium phosphate, calcium propionate,calcium silicate, calcium sorbate, calcium stearate, calcium sulfate,calcium sulfate hemihydrate, canola oil, carbomer, carbon dioxide,carboxymethyl cellulose calcium, carboxymethyl cellulose sodium,β-carotene, carrageenan, castor oil, hydrogenated castor oil, cationicemulsifying wax, cellulose acetate, cellulose acetate phthalate, ethylcellulose, microcrystalline cellulose, powdered cellulose, silicifiedmicrocrystalline cellulose, sodium carboxymethyl cellulose, cetostearylalcohol, cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol,chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidinegluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC),chlorodifluoromethane, chlorofluorocarbons (CFC) chlorophenoxyethanol,chloroxylenol, corn syrup solids, anhydrous citric acid, citric acidmonohydrate, cocoa butter, coloring agents, corn oil, cottonseed oil,cresol, m-cresol, o-cresol, p-cresol, croscarmellose sodium,crospovidone, cyclamic acid, cyclodextrins, dextrates, dextrin,dextrose, dextrose anhydrous, diazolidinyl urea, dibutyl phthalate,dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane(HFC), dimethyl-β-cyclodextrin, cyclodextrin-type compounds such asCaptisol®, dimethyl ether, dimethyl phthalate, dipotassium edentate,disodium edentate, disodium hydrogen phosphate, docusate calcium,docusate potassium, docusate sodium, dodecyl gallate,dodecyltrimethylammonium bromide, edentate calcium disodium, edtic acid,eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate,ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium,ethylparaben sodium, ethyl vanillin, fructose, fructose liquid, fructosemilled, fructose pyrogen-free, powdered fructose, fumaric acid, gelatin,glucose, liquid glucose, glyceride mixtures of saturated vegetable fattyacids, glycerin, glyceryl behenate, glyceryl monooleate, glycerylmonostearate, self-emulsifying glyceryl monostearate, glycerylpalmitostearate, glycine, glycols, glycofurol, guar gum,heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, highfructose syrup, human serum albumin, hydrocarbons (HC), dilutehydrochloric acid, hydrogenated vegetable oil type II, hydroxyethylcellulose, 2-hydroxyethyl-β-cyclodextrin, hydroxypropyl cellulose,low-substituted hydroxypropyl cellulose, 2-hydroxypropyl-β-cyclodextrin,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,imidurea, indigo carmine, ion exchangers, iron oxides, isopropylalcohol, isopropyl myristate, isopropyl palmitate, isotonic saline,kaolin, lactic acid, lactitol, lactose, lanolin, lanolin alcohols,anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesiumcarbonate, normal magnesium carbonate, magnesium carbonate anhydrous,magnesium carbonate hydroxide, magnesium hydroxide, magnesium laurylsulfate, magnesium oxide, magnesium silicate, magnesium stearate,magnesium trisilicate, magnesium trisilicate anhydrous, malic acid,malt, maltitol, maltitol solution, maltodextrin, maltol, maltose,mannitol, medium chain triglycerides, meglumine, menthol,methylcellulose, methyl methacrylate, methyl oleate, methylparaben,methylparaben potassium, methylparaben sodium, microcrystallinecellulose and carboxymethylcellulose sodium, mineral oil, light mineraloil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine,montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin,peanut oil, petrolatum, petrolatum and lanolin alcohols, pharmaceuticalglaze, phenol, liquified phenol, phenoxyethanol, phenoxypropanol,phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, polacrilin, polacrilin potassium, poloxamer,polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates,polyethylene-polyoxypropylene-block polymers, polymethacrylates,polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearates,polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassiumbenzoate, potassium bicarbonate, potassium bisulfite, potassiumchloride, postassium citrate, potassium citrate anhydrous, potassiumhydrogen phosphate, potassium metabisulfite, monobasic potassiumphosphate, potassium propionate, potassium sorbate, povidone, propanol,propionic acid, propylene carbonate, propylene glycol, propylene glycolalginate, propyl gallate, propylparaben, propylparaben potassium,propylparaben sodium, protamine sulfate, rapeseed oil, Ringer'ssolution, saccharin, saccharin ammonium, saccharin calcium, saccharinsodium, safflower oil, saponite, serum proteins, sesame oil, colloidalsilica, colloidal silicon dioxide, sodium alginate, sodium ascorbate,sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride,anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride,sodium cyclamate, sodium edentate, sodium dodecyl sulfate, sodium laurylsulfate, sodium metabisulfite, sodium phosphate, dibasic, sodiumphosphate, monobasic, sodium phosphate, tribasic, anhydrous sodiumpropionate, sodium propionate, sodium sorbate, sodium starch glycolate,sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters(sorbitan fatty esters), sorbitol, sorbitol solution 70%, soybean oil,spermaceti wax, starch, corn starch, potato starch, pregelatinizedstarch, sterilizable maize starch, stearic acid, purified stearic acid,stearyl alcohol, sucrose, sugars, compressible sugar, confectioner'ssugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF,synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane(HFC), theobroma oil, thimerosal, titanium dioxide, alpha tocopherol,tocopheryl acetate, alpha tocopheryl acid succinate, beta-tocopherol,delta-tocopherol, gamma-tocopherol, tragacanth, triacetin, tributylcitrate, triethanolamine, triethyl citrate, trimethyl-β-cyclodextrin,trimethyltetradecylammonium bromide, tris buffer, trisodium edentate,vanillin, type I hydrogenated vegetable oil, water, soft water, hardwater, carbon dioxide-free water, pyrogen-free water, water forinjection, sterile water for inhalation, sterile water for injection,sterile water for irrigation, waxes, anionic emulsifying wax, carnaubawax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax,nonionic emulsifying wax, suppository wax, white wax, yellow wax, whitepetrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zincsalts, zinc stearate, or any excipient in the Handbook of PharmaceuticalExcipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London,UK, 2000), which is incorporated by reference in its entirety.Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin(Mack Publishing Co., Easton, Pa., 1980), which is incorporated byreference in its entirety, discloses various components used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional agent is incompatible with the pharmaceutical compositions,its use in pharmaceutical compositions is contemplated. Supplementaryactive ingredients also can be incorporated into the compositions.

In some embodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration, such as, for example,at least A, wherein A is 0.0001% w/v, 0.001% w/v, 0.01% w/v, 0.1% w/v,1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60%w/v, 70% w/v, 80% w/v, or 90% w/v. In some embodiments, the foregoingcomponent(s) may be present in the pharmaceutical composition at anyconcentration, such as, for example, at most B, wherein B is 90% w/v,80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v,5% w/v, 2% w/v, 1% w/v, 0.1% w/v, 0.001% w/v, or 0.0001%. In otherembodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration range, such as, forexample from about A to about B. In some embodiments, A is 0.0001% and Bis 90%.

In some embodiments, the pharmaceutically acceptable ingredient isselected from the group consisting of a sugar (e.g., glucose, sucrose,trehalose, lactose, fructose, maltose, dextran, glycerin, dextran,mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose,lactulose, maltulose, or iso-maltulose, or combinations of thesesugars), a sugar alcohol (e.g., glycol, glycerol, erythritol, threitol,arabitol, xylitol, ribitol, mannitol, sorbitol, dulcitol, iditol,isomalt, maltitol, lactitol, or glucitol, or combinations of these sugaralcohols), a salt (e.g., sodium chloride), an emulsifier or surfactant(e.g., polysorbates, such as polyoxyethylene 20 sorbitan monooleate, orother block copolymers of ethylene oxide and propylene oxide),lyoprotectants, and mixtures thereof. For example, excipients such assugars or sugar alcohols are present, e.g., in a concentration of about20 mg/mL to about 40 mg/mL, or 25 to 45 mg/mL, such as 35 mg/mL.

The pharmaceutical compositions may be formulated to achieve aphysiologically compatible pH. In some embodiments, the pH of thepharmaceutical composition may be at least 5, at least 5.5, at least 6,at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, atleast 9, at least 9.5, at least 10, or at least 10.5 up to and includingpH 11, depending on the formulation and route of administration, forexample between 4 and 7, or 4.5 and 5.5. In exemplary embodiments, thepharmaceutical compositions may comprise buffering agents to achieve aphysiological compatible pH. The buffering agents may include anycompounds capable of buffering at the desired pH such as, for example,phosphate buffers (e.g., PBS), triethanolamine, Tris, bicine, TAPS,tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES, acetate, citrate,succinate, histidine or other pharmaceutically acceptable buffers. Inexemplary embodiments, the strength of the buffer is at least 0.5 mM, atleast 1 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 30mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, atleast 80 mM, at least 90 mM, at least 100 mM, at least 120 mM, at least150 mM, or at least 200 mM. In some embodiments, the strength of thebuffer is no more than 300 mM (e.g., at most 200 mM, at most 100 mM, atmost 90 mM, at most 80 mM, at most 70 mM, at most 60 mM, at most 50 mM,at most 40 mM, at most 30 mM, at most 20 mM, at most 10 mM, at most 5mM, at most 1 mM). For example, the buffer concentration can be about 2mM to about 100 mM, or about 10 mM to about 50 mM.

Routes of Administration

The following discussion on routes of administration is merely providedto illustrate exemplary embodiments and should not be construed aslimiting the scope in any way.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the analog of the presentdisclosure dissolved in diluents, such as water, saline, or orangejuice; (b) capsules, sachets, tablets, lozenges, and troches, eachcontaining a predetermined amount of the active ingredient, as solids orgranules; (c) powders; (d) suspensions in an appropriate liquid; and (e)suitable emulsions. Liquid formulations may include diluents, such aswater and alcohols, for example, ethanol, benzyl alcohol, and thepolyethylene alcohols, either with or without the addition of apharmaceutically acceptable surfactant. Capsule forms can be of theordinary hard- or soft-shelled gelatin type containing, for example,surfactants, lubricants, and inert fillers, such as lactose, sucrose,calcium phosphate, and corn starch. Tablet forms can include one or moreof lactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise the analog of the presentdisclosure in a flavor, usually sucrose and acacia or tragacanth, aswell as pastilles comprising the analog of the present disclosure in aninert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to, suchexcipients as are known in the art.

The analogs of the disclosure, alone or in combination with othersuitable components, can be delivered via pulmonary administration andcan be made into aerosol formulations to be administered via inhalation.These aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also may be formulated as pharmaceuticals for non-pressuredpreparations, such as in a nebulizer or an atomizer. Such sprayformulations also may be used to spray mucosa. In some embodiments, theanalog is formulated into a powder blend or into microparticles ornanoparticles. Suitable pulmonary formulations are known in the art.See, e.g., Qian et al., Int J Pharm 366: 218-220 (2009); Adjei andGarren, Pharmaceutical Research, 7(6): 565-569 (1990); Kawashima et al.,J Controlled Release 62 (1-2): 279-287 (1999); Liu et al., Pharm Res10(2): 228-232 (1993); International Patent Application Publication Nos.WO 2007/133747 and WO 2007/141411.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The term, “parenteral” means not through the alimentary canal but bysome other route such as subcutaneous, intramuscular, intraspinal, orintravenous. The analog of the present disclosure can be administeredwith a physiologically acceptable diluent in a pharmaceutical carrier,such as a sterile liquid or mixture of liquids, including water, saline,aqueous dextrose and related sugar solutions, an alcohol, such asethanol or hexadecyl alcohol, a glycol, such as propylene glycol orpolyethylene glycol, dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain from about 0.5% toabout 25% by weight of the analog of the present disclosure in solution.Preservatives and buffers may be used. In order to minimize or eliminateirritation at the site of injection, such compositions may contain oneor more nonionic surfactants having a hydrophile-lipophile balance (HLB)of from about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5% to about 15% by weight.Suitable surfactants include polyethylene glycol sorbitan fatty acidesters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986)).

Additionally, the analog of the present disclosures can be made intosuppositories for rectal administration by mixing with a variety ofbases, such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration can be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulas containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the analog of thedisclosure can be formulated as inclusion complexes, such ascyclodextrin inclusion complexes, or liposomes.

Dose

The analogs of the disclosure are believed to be useful in methods oftreating a disease or medical condition in which GIP receptor agonism,GIP/GLP-1 receptor co-agonism, GIP/glucagon receptor co-agonism, orGIP/GLP-1/glucagon receptor triagonism plays a role. For purposes of thedisclosure, the amount or dose of the analog of the present disclosureadministered should be sufficient to effect, e.g., a therapeutic orprophylactic response, in the subject or animal over a reasonable timeframe. For example, the dose of the analog of the present disclosureshould be sufficient to stimulate cAMP secretion from cells as describedherein or sufficient to decrease blood glucose levels, fat levels, foodintake levels, or body weight of a mammal, in a period of from about 1to 4 minutes, 1 to 4 hours or 1 to 4 weeks or longer, e.g., 5 to 20 ormore weeks, from the time of administration. In exemplary embodiments,the time period could be even longer. The dose will be determined by theefficacy of the particular analog of the present disclosure and thecondition of the animal (e.g., human), as well as the body weight of theanimal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes herein, an assay, which comprises comparing the extent towhich blood glucose levels are lowered upon administration of a givendose of the analog of the present disclosure to a mammal among a set ofmammals of which is each given a different dose of the analog, could beused to determine a starting dose to be administered to a mammal. Theextent to which blood glucose levels are lowered upon administration ofa certain dose can be assayed by methods known in the art, including,for instance, the methods described herein as Example 11.

The dose of the analog of the present disclosure also will be determinedby the existence, nature and extent of any adverse side effects thatmight accompany the administration of a particular analog of the presentdisclosure. Typically, the attending physician will decide the dosage ofthe analog of the present disclosure with which to treat each individualpatient, taking into consideration a variety of factors, such as age,body weight, general health, diet, sex, analog of the present disclosureto be administered, route of administration, and the severity of thecondition being treated. By way of example and not intending to limitthe invention, the dose of the analog of the present disclosure can beabout 0.0001 to about 1 g/kg body weight of the subject beingtreated/day, from about 0.0001 to about 0.001 g/kg body weight/day, orabout 0.01 mg to about 1 g/kg body weight/day. In exemplary embodiments,the dose can be a total weekly dose of about 1 mg to about 40 mg, orabout 4 mg to about 30 mg, or about 4 to about 20 mg, or about 10 toabout 20 mg, or about 12 mg to about 30 mg.

In some embodiments, the pharmaceutical composition comprises any of theanalogs disclosed herein at a purity level suitable for administrationto a patient. In some embodiments, the analog has a purity level of atleast about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98% or about 99%, and a pharmaceuticallyacceptable diluent, carrier or excipient. The pharmaceutical compositionin some aspects comprise the analog of the present disclosure at aconcentration of at least A, wherein A is about 0.001 mg/ml, about 0.01mg/ml, 0 about 1 mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml,about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 11 mg/ml,about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml, about 20 mg/ml,about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24 mg/ml, about 25mg/ml or higher. In some embodiments, the pharmaceutical compositioncomprises the analog at a concentration of at most B, wherein B is about30 mg/ml, about 25 mg/ml, about 24 mg/ml, about 23, mg/ml, about 22mg/ml, about 21 mg/ml, about 20 mg/ml, about 19 mg/ml, about 18 mg/ml,about 17 mg/ml, about 16 mg/ml, about 15 mg/ml, about 14 mg/ml, about 13mg/ml, about 12 mg/ml, about 11 mg/ml, about 10 mg/ml, about 9 mg/ml,about 8 mg/ml, about 7 mg/ml, about 6 mg/ml, about 5 mg/ml, about 4mg/ml, about 3 mg/ml, about 2 mg/ml, about 1 mg/ml, or about 0.1 mg/ml.In some embodiments, the compositions may contain an analog at aconcentration range of A to B mg/ml, for example, about 0.001 to about30.0 mg/ml.

Targeted Forms

One of ordinary skill in the art will readily appreciate that theanalogs of the disclosure can be modified in any number of ways, suchthat the therapeutic or prophylactic efficacy of the analog of thepresent disclosures is increased through the modification. For instance,the analog of the present disclosure can be conjugated either directlyor indirectly through a linker to a targeting moiety. The practice ofconjugating compounds, e.g., glucagon analogs described herein, totargeting moieties is known in the art. See, for instance, Wadhwa etal., J Drug Targeting, 3, 111-127 (1995) and U.S. Pat. No. 5,087,616.The term “targeting moiety” as used herein, refers to any molecule oragent that specifically recognizes and binds to a cell-surface receptor,such that the targeting moiety directs the delivery of the analog of thepresent disclosures to a population of cells on which surface thereceptor (the glucagon receptor, the GLP-1 receptor) is expressed.Targeting moieties include, but are not limited to, antibodies, orfragments thereof, peptides, hormones, growth factors, cytokines, andany other natural or non-natural ligands, which bind to cell surfacereceptors (e.g., Epithelial Growth Factor Receptor (EGFR), T-cellreceptor (TCR), B-cell receptor (BCR), CD28, Platelet-derived GrowthFactor Receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.).As used herein a “linker” is a bond, molecule or group of molecules thatbinds two separate entities to one another. Linkers may provide foroptimal spacing of the two entities or may further supply a labilelinkage that allows the two entities to be separated from each other.Labile linkages include photocleavable groups, acid-labile moieties,base-labile moieties and enzyme-cleavable groups. The term “linker” insome embodiments refers to any agent or molecule that bridges the analogof the present disclosures to the targeting moiety. One of ordinaryskill in the art recognizes that sites on the analog of the presentdisclosures, which are not necessary for the function of the analog ofthe present disclosures, are ideal sites for attaching a linker and/or atargeting moiety, provided that the linker and/or targeting moiety, onceattached to the analog of the present disclosures, do(es) not interferewith the function of the analog of the present disclosures, i.e., theability to stimulate cAMP secretion from cells, to treat diabetes orobesity.

Controlled Release Formulations

Alternatively, the glucagon analogs described herein can be modifiedinto a depot form, such that the manner in which the analog of thepresent disclosures is released into the body to which it isadministered is controlled with respect to time and location within thebody (see, for example, U.S. Pat. No. 4,450,150). Depot forms of analogof the present disclosures can be, for example, an implantablecomposition comprising the analog of the present disclosures and aporous or non-porous material, such as a polymer, wherein the analog ofthe present disclosures is encapsulated by or diffused throughout thematerial and/or degradation of the non-porous material. The depot isthen implanted into the desired location within the body and the analogof the present disclosures are released from the implant at apredetermined rate.

The pharmaceutical composition in exemplary aspects is modified to haveany type of in vivo release profile. In some aspects, the pharmaceuticalcomposition is an immediate release, controlled release, sustainedrelease, extended release, delayed release, or bi-phasic releaseformulation. Methods of formulating peptides for controlled release areknown in the art. See, for example, Qian et al., J Pharm 374: 46-52(2009) and International Patent Application Publication Nos. WO2008/130158, WO2004/033036; WO2000/032218; and WO 1999/040942.

The instant compositions may further comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect. The disclosed pharmaceutical formulations may be administeredaccording to any regime including, for example, daily (1 time per day, 2times per day, 3 times per day, 4 times per day, 5 times per day, 6times per day), three times a week, twice a week, every two days, everythree days, every four days, every five days, every six days, weekly,bi-weekly, every three weeks, monthly, or bi-monthly.

Combinations

The glucagon analogs described herein may be administered alone or incombination with other therapeutic agents which aim to treat or preventany of the diseases or medical conditions described herein. For example,the glucagon analogs described herein may be co-administered with(simultaneously or sequentially) an anti-diabetic or anti-obesity agent.Anti-diabetic agents known in the art or under investigation includeinsulin, leptin, Peptide YY (PYY), Pancreatic Peptide (PP), fibroblastgrowth factor 21 (FGF21), Y2Y4 receptor agonists, sulfonylureas, such astolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase),chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta,Micronase, Glynase), glimepiride (Amaryl), or gliclazide (Diamicron);meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix);biguanides such as metformin (Glucophage) or phenformin;thiazolidinediones such as rosiglitazone (Avandia), pioglitazone(Actos), or troglitazone (Rezulin), or other PPARγ inhibitors; alphaglucosidase inhibitors that inhibit carbohydrate digestion, such asmiglitol (Glyset), acarbose (Precose/Glucobay); exenatide (Byetta) orpramlintide; Dipeptidyl peptidase-4 (DPP-4) inhibitors such asvildagliptin or sitagliptin; SGLT (sodium-dependent glucosetransporter 1) inhibitors; glucokinase activators (GKA); glucagonreceptor antagonists (GRA); or FBPase (fructose 1,6-bisphosphatase)inhibitors.

Anti-obesity agents known in the art or under investigation includeappetite suppressants, including phenethylamine type stimulants,phentermine (optionally with fenfluramine or dexfenfluramine),diethylpropion (Tenuate®), phendimetrazine (Prelu-2®, Bontril®),benzphetamine (Didrex®), sibutramine (Meridia®, Reductil®); rimonabant(Acomplia®), other cannabinoid receptor antagonists; oxyntomodulin;fluoxetine hydrochloride (Prozac); Qnexa (topiramate and phentermine),Excalia (bupropion and zonisamide) or Contrave (bupropion andnaltrexone); or lipase inhibitors, similar to XENICAL (Orlistat) orCetilistat (also known as ATL-962), or GT 389-255.

The peptides described herein in some embodiments are co-administeredwith an agent for treatment of non-alcoholic fatty liver disease orNASH. Agents used to treat non-alcoholic fatty liver disease includeursodeoxycholic acid (a.k.a., Actigall, URSO, and Ursodiol), Metformin(Glucophage), rosiglitazone (Avandia), Clofibrate, Gemfibrozil,Polymixin B, and Betaine.

The peptides described herein in some embodiments are co-administeredwith an agent for treatment of a neurodegenerative disease, e.g.,Parkinson's Disease. Anti-Parkinson's Disease agents are furthermoreknown in the art and include, but not limited to, levodopa, carbidopa,anticholinergics, bromocriptine, pramipexole, and ropinirole,amantadine, and rasagiline.

In view of the foregoing, the invention further provides pharmaceuticalcompositions and kits additionally comprising one of these othertherapeutic agents. The additional therapeutic agent may be administeredsimultaneously or sequentially with the analog of the presentdisclosure. In some aspects, the analog is administered before theadditional therapeutic agent, while in other aspects, the analog isadministered after the additional therapeutic agent.

Uses

Based on the information provided for the first time herein, it iscontemplated that the compositions (e.g., related pharmaceuticalcompositions) of the present disclosures are useful for treatment of adisease or medical condition, in which e.g., the lack of activity at theGIP receptor, the GLP-1 receptor, or at both receptors, is a factor inthe onset and/or progression of the disease or medical condition.Accordingly, the present disclosures provides a method of treating orpreventing a disease or medical condition in a patient, wherein thedisease or medical condition is a disease of medical condition in whicha lack of GIP receptor activation and/or GLP-1 receptor activation isassociated with the onset and/or progression of the disease of medicalcondition. The method comprises providing to the patient a compositionor conjugate in accordance with any of those described herein in anamount effective to treat or prevent the disease or medical condition.

In some embodiments, the disease or medical condition is metabolicsyndrome. Metabolic Syndrome, also known as metabolic syndrome X,insulin resistance syndrome or Reaven's syndrome, is a disorder thataffects over 50 million Americans. Metabolic Syndrome is typicallycharacterized by a clustering of at least three or more of the followingrisk factors: (1) abdominal obesity (excessive fat tissue in and aroundthe abdomen), (2) atherogenic dyslipidemia (blood fat disordersincluding high triglycerides, low HDL cholesterol and high LDLcholesterol that enhance the accumulation of plaque in the arterywalls), (3) elevated blood pressure, (4) insulin resistance or glucoseintolerance, (5) prothrombotic state (e.g., high fibrinogen orplasminogen activator inhibitor-1 in blood), and (6) pro-inflammatorystate (e.g., elevated C-reactive protein in blood). Other risk factorsmay include aging, hormonal imbalance and genetic predisposition.

Metabolic Syndrome is associated with an increased the risk of coronaryheart disease and other disorders related to the accumulation ofvascular plaque, such as stroke and peripheral vascular disease,referred to as atherosclerotic cardiovascular disease (ASCVD). Patientswith Metabolic Syndrome may progress from an insulin resistant state inits early stages to full blown type II diabetes with further increasingrisk of ASCVD. Without intending to be bound by any particular theory,the relationship between insulin resistance, Metabolic Syndrome andvascular disease may involve one or more concurrent pathogenicmechanisms including impaired insulin-stimulated vasodilation, insulinresistance-associated reduction in NO availability due to enhancedoxidative stress, and abnormalities in adipocyte-derived hormones suchas adiponectin (Lteif and Mather, Can. J. Cardiol. 20 (suppl. B):66B-76B(2004)).

According to the 2001 National Cholesterol Education Program AdultTreatment Panel (ATP III), any three of the following traits in the sameindividual meet the criteria for Metabolic Syndrome: (a) abdominalobesity (a waist circumference over 102 cm in men and over 88 cm inwomen); (b) serum triglycerides (150 mg/dl or above); (c) HDLcholesterol (40 mg/dl or lower in men and 50 mg/dl or lower in women);(d) blood pressure (130/85 or more); and (e) fasting blood glucose (110mg/dl or above). According to the World Health Organization (WHO), anindividual having high insulin levels (an elevated fasting blood glucoseor an elevated post meal glucose alone) with at least two of thefollowing criteria meets the criteria for Metabolic Syndrome: (a)abdominal obesity (waist to hip ratio of greater than 0.9, a body massindex of at least 30 kg/m2, or a waist measurement over 37 inches); (b)cholesterol panel showing a triglyceride level of at least 150 mg/dl oran HDL cholesterol lower than 35 mg/dl; (c) blood pressure of 140/90 ormore, or on treatment for high blood pressure). (Mathur, Ruchi,“Metabolic Syndrome,” ed. Shiel, Jr., William C., MedicineNet.com, May11, 2009).

For purposes herein, if an individual meets the criteria of either orboth of the criteria set forth by the 2001 National CholesterolEducation Program Adult Treatment Panel or the WHO, that individual isconsidered as afflicted with Metabolic Syndrome.

Without being bound to any particular theory, compositions andconjugates described herein are useful for treating Metabolic Syndrome.Accordingly, the invention provides a method of preventing or treatingMetabolic Syndrome, or reducing one, two, three or more risk factorsthereof, in a subject, comprising providing to the subject a compositiondescribed herein in an amount effective to prevent or treat MetabolicSyndrome, or the risk factor thereof.

In some embodiments, the method treats a hyperglycemic medicalcondition. In exemplary aspects, the hyperglycemic medical condition isdiabetes, diabetes mellitus type I, diabetes mellitus type II, orgestational diabetes, either insulin-dependent or non-insulin-dependent.In some aspects, the method treats the hyperglycemic medical conditionby reducing one or more complications of diabetes including nephropathy,retinopathy and vascular disease.

In some aspects, the disease or medical condition is obesity. In someaspects, the obesity is drug-induced obesity. In some aspects, themethod treats obesity by preventing or reducing weight gain orincreasing weight loss in the patient. In some aspects, the methodtreats obesity by reducing appetite, decreasing food intake, loweringthe levels of fat in the patient, or decreasing the rate of movement offood through the gastrointestinal system.

Because obesity is associated with the onset or progression of otherdiseases, the methods of treating obesity are further useful in methodsof reducing complications associated with obesity including vasculardisease (coronary artery disease, stroke, peripheral vascular disease,ischemia reperfusion, etc.), hypertension, onset of diabetes type II,hyperlipidemia and musculoskeletal diseases. The present disclosuresaccordingly provides methods of treating or preventing theseobesity-associated complications.

In some embodiments, the disease or medical condition is Nonalcoholicfatty liver disease (NAFLD). NAFLD refers to a wide spectrum of liverdisease ranging from simple fatty liver (steatosis), to nonalcoholicsteatohepatitis (NASH), to cirrhosis (irreversible, advanced scarring ofthe liver). All of the stages of NAFLD have in common the accumulationof fat (fatty infiltration) in the liver cells (hepatocytes). Simplefatty liver is the abnormal accumulation of a certain type of fat,triglyceride, in the liver cells with no inflammation or scarring. InNASH, the fat accumulation is associated with varying degrees ofinflammation (hepatitis) and scarring (fibrosis) of the liver. Theinflammatory cells can destroy the liver cells (hepatocellularnecrosis). In the terms “steatohepatitis” and “steatonecrosis”, steatorefers to fatty infiltration, hepatitis refers to inflammation in theliver, and necrosis refers to destroyed liver cells. NASH can ultimatelylead to scarring of the liver (fibrosis) and then irreversible, advancedscarring (cirrhosis). Cirrhosis that is caused by NASH is the last andmost severe stage in the NAFLD spectrum. (Mendler, Michel, “Fatty Liver:Nonalcoholic Fatty Liver Disease (NAFLD) and NonalcoholicSteatohepatitis (NASH),” ed. Schoenfield, Leslie J., MedicineNet.com,Aug. 29, 2005).

Alcoholic Liver Disease, or Alcohol-Induced Liver Disease, encompassesthree pathologically distinct liver diseases related to or caused by theexcessive consumption of alcohol: fatty liver (steatosis), chronic oracute hepatitis, and cirrhosis. Alcoholic hepatitis can range from amild hepatitis, with abnormal laboratory tests being the only indicationof disease, to severe liver dysfunction with complications such asjaundice (yellow skin caused by bilirubin retention), hepaticencephalopathy (neurological dysfunction caused by liver failure),ascites (fluid accumulation in the abdomen), bleeding esophageal varices(varicose veins in the esophagus), abnormal blood clotting and coma.Histologically, alcoholic hepatitis has a characteristic appearance withballooning degeneration of hepatocytes, inflammation with neutrophilsand sometimes Mallory bodies (abnormal aggregations of cellularintermediate filament proteins). Cirrhosis is characterized anatomicallyby widespread nodules in the liver combined with fibrosis. (Worman,Howard J., “Alcoholic Liver Disease”, Columbia University Medical Centerwebsite).

Without being bound to any particular theory, the compositions andconjugates described herein are useful for the treatment of AlcoholicLiver Disease, NAFLD, or any stage thereof, including, for example,steatosis, steatohepatitis, hepatitis, hepatic inflammation, NASH,cirrhosis, or complications thereof. Accordingly, the presentdisclosures provides a method of preventing or treating Alcoholic LiverDisease, NAFLD, or any stage thereof, in a subject comprising providingto a subject a composition described herein in an amount effective toprevent or treat Alcoholic Liver Disease, NAFLD, or the stage thereof.Such treatment methods include reduction in one, two, three or more ofthe following: liver fat content, incidence or progression of cirrhosis,incidence of hepatocellular carcinoma, signs of inflammation, e.g.,abnormal hepatic enzyme levels (e.g., aspartate aminotransferase ASTand/or alanine aminotransferase ALT, or LDH), elevated serum ferritin,elevated serum bilirubin, and/or signs of fibrosis, e.g., elevatedTGF-beta levels. In exemplary embodiments, the compositions are usedtreat patients who have progressed beyond simple fatty liver (steatosis)and exhibit signs of inflammation or hepatitis. Such methods may result,for example, in reduction of AST and/or ALT levels.

GLP-1 and exendin-4 have been shown to have some neuroprotective effect.The present disclosures also provides uses of the compositions describedherein in treating neurodegenerative diseases, including but not limitedto Alzheimer's disease, Parkinson's disease, Multiple Sclerosis,Amylotrophic Lateral Sclerosis, other demyelination related disorders,senile dementia, subcortical dementia, arteriosclerotic dementia,AIDS-associated dementia, or other dementias, a central nervous systemcancer, traumatic brain injury, spinal cord injury, stroke or cerebralischemia, cerebral vasculitis, epilepsy, Huntington's disease,Tourette's syndrome, Guillain Barre syndrome, Wilson disease, Pick'sdisease, neuroinflammatory disorders, encephalitis, encephalomyelitis ormeningitis of viral, fungal or bacterial origin, or other centralnervous system infections, prion diseases, cerebellar ataxias,cerebellar degeneration, spinocerebellar degeneration syndromes,Friedreichs ataxia, ataxia telangiectasia, spinal dysmyotrophy,progressive supranuclear palsy, dystonia, muscle spasticity, tremor,retinitis pigmentosa, striatonigral degeneration, mitochondrialencephalo-myopathies, neuronal ceroid lipofuscinosis, hepaticencephalopathies, renal encephalopathies, metabolic encephalopathies,toxin-induced encephalopathies, and radiation-induced brain damage.

In some embodiments, the compositions are used in conjunction withparenteral administration of nutrients to non-diabetic patients in ahospital setting, e.g., to patients receiving parenteral nutrition ortotal parenteral nutrition. Nonlimiting examples include surgerypatients, patients in comas, patients with digestive tract illness, or anonfunctional gastrointestinal tract (e.g. due to surgical removal,blockage or impaired absorptive capacity, Crohn's disease, ulcerativecolitis, gastrointestinal tract obstruction, gastrointestinal tractfistula, acute pancreatitis, ischemic bowel, major gastrointestinalsurgery, certain congenital gastrointestinal tract anomalies, prolongeddiarrhea, or short bowel syndrome due to surgery, patients in shock, andpatients undergoing healing processes often receive parenteraladministration of carbohydrates along with various combinations oflipids, electrolytes, minerals, vitamins and amino acids. Thecompositions comprising the GIP agonist peptide and glucagon antagonistpeptide, as described herein, and the parenteral nutrition compositioncan be administered at the same time, at different times, before, orafter each other, provided that the composition is exerting the desiredbiological effect at the time that the parenteral nutrition compositionis being digested. For example, the parenteral nutrition may beadministered, 1, 2 or 3 times per day, while the composition isadministered once every other day, three times a week, two times a week,once a week, once every 2 weeks, once every 3 weeks, or once a month.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. For example, as used herein the term “treating diabetes” willrefer in general to altering glucose blood levels in the direction ofnormal levels and may include increasing or decreasing blood glucoselevels depending on a given situation.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a glucagon peptide refers to a nontoxic but sufficient amountof the peptide to provide the desired effect. For example one desiredeffect would be the prevention or treatment of hypoglycemia, asmeasured, for example, by an increase in blood glucose level. Analternative desired effect for the glucagon peptides of the presentdisclosure would include treating hyperglycemia, e.g., as measured by achange in blood glucose level closer to normal, or inducing weightloss/preventing weight gain, e.g., as measured by reduction in bodyweight, or preventing or reducing an increase in body weight, ornormalizing body fat distribution. The amount that is “effective” willvary from subject to subject, depending on the age and general conditionof the individual, mode of administration, and the like. Thus, it is notalways possible to specify an exact “effective amount.” However, anappropriate “effective” amount in any individual case may be determinedby one of ordinary skill in the art using routine experimentation.

Subjects

With regard to the above methods of treatment, the patient is any host.In some embodiments, the host is a mammal. As used herein, the term“mammal” refers to any vertebrate animal of the mammalia class,including, but not limited to, any of the monotreme, marsupial, andplacental taxas. In some embodiments, the mammal is one of the mammalsof the order Rodentia, such as mice and hamsters, and mammals of theorder Logomorpha, such as rabbits. In exemplary embodiments, the mammalsare from the order Carnivora, including Felines (cats) and Canines(dogs). In exemplary embodiments, the mammals are from the orderArtiodactyla, including Bovines (cows) and S wines (pigs) or of theorder Perssodactyla, including Equines (horses). In some instances, themammals are of the order Primates, Ceboids, or Simoids (monkeys) or ofthe order Anthropoids (humans and apes). In particular embodiments, themammal is a human.

Kits

The glucagon analogs of the present disclosure can be provided inaccordance with one embodiment as part of a kit. Accordingly, in someembodiments, a kit for administering a glucagon analog to a patient inneed thereof is provided wherein the kit comprises a glucagon analog asdescribed herein.

In one embodiment the kit is provided with a device for administeringthe glucagon analog to a subject. The device in some aspects is asyringe needle, pen device, jet injector or other needle-free injector.The kit may alternatively or in addition include one or more containers,e.g., vials, tubes, bottles, single or multi-chambered pre-filledsyringes, cartridges, infusion pumps (external or implantable), jetinjectors, pre-filled pen devices and the like, optionally containingthe glucagon analog in a lyophilized form or in an aqueous solution. Thekits in some embodiments comprise instructions for use. In accordancewith one embodiment the device of the kit is an aerosol dispensingdevice, wherein the composition is prepackaged within the aerosoldevice. In another embodiment the kit comprises a syringe and a needle,and in one embodiment the sterile glucagon composition is prepackagedwithin the syringe.

The kits in some embodiments comprise instructions for use. Theinstructions in some aspects include instructions for use in accordancewith any of the methods described herein. The instructions mayadditionally include instructions for maintaining a healthy diet and/ora physical exercise program. The instructions may be in the form of apaper pamphlet, or in electronic form, e.g., a computer readable storagedevice comprising the instructions.

The following examples are given merely to illustrate the presentinvention and not in any way to limit its scope.

EXAMPLES Example 1

The following provides exemplary methods of synthesizing the peptideanalogs of the present disclosures.

Synthesis of Peptide Fragments of Glucagon

Materials:

All peptides described herein in the EXAMPLES were amidated unlessspecified otherwise.

MBHA resin (4-methylbenzhydrylamine polystyrene resin was used duringpeptide synthesis. MBHA resin, 100-180 mesh, 1% DVB cross-linkedpolystyrene; loading of 0.7-1.0 mmol/g), Boc-protected and Fmocprotected amino acids were purchased from Midwest Biotech. The solidphase peptide syntheses using Boc-protected amino acids were performedon an Applied Biosystem 430A Peptide Synthesizer. Fmoc protected aminoacid synthesis was performed using the Applied Biosystems Model 433Peptide Synthesizer.

Peptide Synthesis (Boc Amino Acids/HF Cleavage):

Synthesis of these analogs was performed on the Applied Biosystem Model430A Peptide Synthesizer. Synthetic peptides were constructed bysequential addition of amino acids to a cartridge containing 2 mmol ofBoc protected amino acid. Specifically, the synthesis was carried outusing Boc DEPBT-activated single couplings. At the end of the couplingstep, the peptidyl-resin was treated with TFA to remove the N-terminalBoc protecting group. It was washed repeatedly with dimethylformamide(DMF) and this repetitive cycle was repeated for the desired number ofcoupling steps. After the assembly, the sidechain protection, Fmoc, wasremoved by 20% piperidine treatment and acylation was conducted usingDIC. The peptidyl-resin at the end of the entire synthesis was dried byusing dichloromethane (DCM), and the peptide was cleaved from the resinwith anhydrous HF.

For the lactamization, orthogonal protecting groups were selected forGlu and Lys (e.g., Glu(Fm), Lys(Fmoc)). After removal of the protectinggroups and before HF cleavage, cyclization was performed as describedpreviously (see, e.g., International Patent Application Publication No.WO2008/101017).

HF Treatment of the Peptidyl-Resin

The peptidyl-resin was treated with anhydrous hydrogen fluoride (HF),and this typically yielded approximately 350 mg (˜50% yield) of a crudedeprotected-peptide. Specifically, the peptidyl-resin (30 mg to 200 mg)was placed in the HF reaction vessel for cleavage. 500 μL of p-cresolwas added to the vessel as a carbonium ion scavenger. The vessel wasattached to the HF system and submerged in the methanol/dry ice mixture.The vessel was evacuated with a vacuum pump and 10 ml of HF wasdistilled to the reaction vessel. This reaction mixture of thepeptidyl-resin and the HF was stirred for one hour at 0° C., after whicha vacuum was established and the HF was quickly evacuated (10-15 min).The vessel was removed carefully and filled with approximately 35 ml ofether to precipitate the peptide and to extract the p-cresol and smallmolecule organic protecting groups resulting from HF treatment. Thismixture was filtered utilizing a teflon filter and repeated twice toremove all excess cresol. This filtrate was discarded. The precipitatedpeptide dissolves in approximately 20 ml of 10% acetic acid (aq). Thisfiltrate, which contained the desired peptide, was collected andlyophilized.

An analytical HPLC analysis of the crude solubilized peptide wasconducted under the following conditions [4.6×30 mm Xterra C8, 1.50mL/min, 220 nm, A buffer 0.1% trifluoroacetic acid (TFA)/10%acrylonitrile (CAN), B buffer 0.1% TFA/100% ACN, gradient 5-95% B over15 minutes]. The extract was diluted twofold with water and loaded ontoa 2.2×25 cm Vydac C4 preparative reverse phase column and eluted usingan acetonitrile gradient on a Waters HPLC system (A buffer of 0.1%TFA/10% ACN, B buffer of 0.1% TFA/10% ACN and a gradient of 0-100% Bover 120 minutes at a flow of 15.00 ml/min. HPLC analysis of thepurified peptide demonstrated greater than 95% purity and electrosprayionization mass spectral analysis was used to confirm the identity ofthe peptide.

Peptide Acylation

Acylated peptides were prepared as follows. Peptides were synthesized ona solid support resin using either a CS Bio 4886 Peptide Synthesizer orApplied Biosystems 430A Peptide Synthesizer. In situ neutralizationchemistry was used as described by Schnolzer et al., Int. J. PeptideProtein Res. 40: 180-193 (1992). For acylated peptides, the target aminoacid residue to be acylated (e.g., position ten, relative to the aminoacid position numbering of SEQ ID NO: 3) was substituted with an Nε-FMOC lysine residue. Treatment of the completed N-terminally BOCprotected peptide with 20% piperidine in DMF for 30 minutes removedFMOC/formyl groups. Coupling to the free ε-amino Lys residue wasachieved by coupling a ten-fold molar excess of either an FMOC-protectedspacer amino acid (ex. FMOC-Glu-OtBu) or acyl chain (ex.CH₃(CH₂)₁₄—COOH) and PyBOP or DEPBT coupling reagent inDMF/N,N-diisopropylethylamine (DIEA). Subsequent removal of the spaceramino acid's FMOC group is followed by repetition of coupling with anacyl chain. Final treatment with 100% TFA resulted in removal of anyside chain protecting groups and the N-terminal BOC group. Peptideresins were neutralized with 5% DIEA/DMF, dried, and then cleaved fromthe support using HF/p-cresol, 95:5, at 0° C. for one hour. Followingether extraction, a 5% acetic acid (HOAc) solution was used to solvatethe crude peptide. A sample of the solution was then verified to containthe correct molecular weight peptide by ESI-MS. Correct peptides werepurified by RP-HPLC using a linear gradient of 10% acetonitrile(CH3CN)/0.1% TFA to 0.1% TFA in 100% CH3CN. A Vydac C18 22 mm×250 mmprotein column was used for the purification. Acylated peptide analogsgenerally completed elution by a buffer ratio of 20:80. Portions werepooled together and checked for purity on an analytical RP-HPLC. Purefractions were lyophilized yielding white, solid peptides.

Below is an exemplary acylated Lys residue wherein the acyl group isattached via a γ-Glu-γ-Glu dipeptide spacer.

wherein n is 1 to 29.

If a peptide comprised a lactam bridge and target residues to beacylated, acylation is carried out as described above upon addition ofthat amino acid to the peptide backbone.

Dual acylations or di-acylations are prepared as follows. Peptides aresynthesized on a solid support resin using either a CS Bio 4886 PeptideSynthesizer or Applied Biosystems 430A Peptide Synthesizer. In situneutralization chemistry is used as described by Schnolzer et al., Int.J. Peptide Protein Res. 40: 180-193 (1992). For two site double acylatedpeptides, the target amino acid residues to be acylated (e.g., positionten and 40, relative to the amino acid position numbering of SEQ ID NO:3) are substituted with an N ε-FMOC lysine residue. Treatment of thecompleted N-terminally BOC protected peptide with 20% piperidine in DMFfor 30 minutes removes FMOC/formyl groups. Coupling to the free ε-aminoLys residue is achieved by coupling a ten-fold molar excess of either anFMOC-protected spacer amino acid (ex. FMOC-Glu-OtBu) or acyl chain (ex.CH₃(CH₂)₁₄—COOH) and PyBOP or DEPBT coupling reagent in DMF/DIEA.Subsequent removal of the spacer amino acid's FMOC group is followed byrepetition of coupling with an acyl chain. Final treatment with 100% TFAresults in removal of any side chain protecting groups and theN-terminal BOC group. Peptide resins are neutralized with 5% DIEA/DMF,dried, and then are cleaved from the support using HF/p-cresol, 95:5, at0° C. for one hour. Following ether extraction, a 5% HOAc solution isused to solvate the crude peptide. A sample of the solution is thenverified to contain the correct molecular weight peptide by ESI-MS.Correct peptides are purified by RP-HPLC using a linear gradient of 10%CH3CN/0.1% TFA to 0.1% TFA in 100% CH3CN. A Vydac C18 22 mm×250 mmprotein column was used for the purification. Acylated peptide analogsgenerally complete elution by a buffer ratio of 20:80. Portions arepooled together and checked for purity on an analytical RP-HPLC. Purefractions are lyophilized yielding white, solid peptides.

One site branch double acylations are prepared as follows. Peptides aresynthesized on a solid support resin using either a CS Bio 4886 PeptideSynthesizer or Applied Biosystems 430A Peptide Synthesizer. In situneutralization chemistry is used as described by Schnolzer et al., Int.J. Peptide Protein Res. 40: 180-193 (1992). For acylated peptides, thetarget amino acid residue to be acylated (e.g., position ten relative tothe amino acid position numbering of SEQ ID NO: 3) is substituted withan N ε-FMOC lysine residue. Treatment of the completed N-terminally BOCprotected peptide with 20% piperidine in DMF for 30 minutes removesFMOC/formyl groups. Coupling to a Lys residue through the C-terminus isachieved by coupling a ten-fold molar excess of an ε-amino and α-aminoFMOC-protected spacer amino acid (ex. FMOC-Lys(FMOC)-OH) and PyBOP orDEPBT coupling reagent in DMF/DIEA. Subsequent removal of the spaceramino acid's FMOC groups is followed by coupling each of the ε-amino andα-amino groups with an acyl chain. Final treatment with 100% TFA resultsin removal of any side chain protecting groups and the N-terminal BOCgroup. Peptide resins are neutralized with 5% DIEA/DMF, dried, and thenare cleaved from the support using HF/p-cresol, 95:5, at 0° C. for onehour. Following ether extraction, a 5% HOAc solution is used to solvatethe crude peptide. A sample of the solution is then verified to containthe correct molecular weight peptide by ESI-MS. Correct peptides arepurified by RP-HPLC using a linear gradient of 10% CH3CN/0.1% TFA to0.1% TFA in 100% CH3CN. A Vydac C18 22 mm×250 mm protein column was usedfor the purification. Acylated peptide analogs generally completeelution by a buffer ratio of 20:80. Portions are pooled together andchecked for purity on an analytical RP-HPLC. Pure fractions werelyophilized yielding white, solid peptides.

One site linear double acylations are prepared as follows. Peptides aresynthesized on a solid support resin using either a CS Bio 4886 PeptideSynthesizer or Applied Biosystems 430A Peptide Synthesizer. In situneutralization chemistry is used as described by Schnolzer et al., Int.J. Peptide Protein Res. 40: 180-193 (1992). For acylated peptides, thetarget amino acid residue to be acylated (e.g., position ten, relativeto the amino acid position numbering of SEQ ID NO: 3) is substitutedwith an N ε-FMOC lysine residue. Treatment of the completed N-terminallyBOC protected peptide with 20% piperidine in DMF for 30 minutes removesFMOC/formyl groups. Coupling to the free ε-amino Lys residue is achievedby coupling a ten-fold molar excess of either an FMOC-protected spaceramino acid (ex. FMOC-Glu-OtBu) or acyl chain (ex. CH₃(CH₂)₁₄—COOH) andPyBOP or DEPBT coupling reagent in DMF/DIEA. Subsequent removal of thespacer amino acid's FMOC group is followed by coupling with an acylchain functionalized at the end of the fatty acid tail with a protectedamino acid such a as FmocNH—(CH₂)₁₁—COOH. The resulting single acylatedamino acid is treated with 20% piperidine in DMF to remove the FMOCprotecting group, followed by coupling with an acyl chain. Finaltreatment with 100% TFA results in the removal of any side chainprotecting groups and the N-terminal BOC group. Peptide resins areneutralized with 5% DIEA/DMF, dried, and then cleaved from the supportusing HF/p-cresol, 95:5, at 0° C. for one hour. Following etherextraction, a 5% HOAc solution is used to solvate the crude peptide. Asample of the solution is then verified to contain the correct molecularweight peptide by ESI-MS. Correct peptides are purified by RP-HPLC usinga linear gradient of 10% CH3CN/0.1% TFA to 0.1% TFA in 100% CH3CN. AVydac C18 22 mm×250 mm protein column is used for the purification.Acylated peptide analogs generally complete elution by a buffer ratio of20:80. Portions are pooled together and checked for purity on ananalytical RP-HPLC. Pure fractions are lyophilized yielding white, solidpeptides.

In the instances of two site or single site double acylations, the twoacyl chains to be coupled to the peptide can be the same or different.In the case of two different acyl chains, the target amino acid(s) to beacylated are substituted with two different protecting groups. Forexample an N ε-FMOC lysine residue and an N ε-ivDde lysine residue (twosite double acylation) or an FMOC Lys (ivDde)-OH (single site doubleacylation, 852082 Novabiochem). Treatment of the completed N-terminallyBOC protected peptide with 20% piperidine in DMF for 30 minutes removedFMOC/formyl groups. The free ε-amino Lys residue is coupled with eitheran acyl group or spacer amino acid followed by coupling to an acylgroup. The resulting single acylated peptide is treated with 2%hydrazine/DMF to remove the (ivDde) protecting group. The free amino Lysresidue is coupled with either an acyl group or a spacer amino acidfollowed by coupling to an acy group. The double acylated peptide isisolated as described above.

An example synthesis of a dual acylated peptide (Glucagon Aib2 E16 A18L27 D28 Cex K40(C16γE-K-γEC12)G41-amide, having the amino acid sequenceof SEQ ID NO: 211) is shown in FIG. 5B and is further described below:

0.28 gm (0.2 mmole) mbha-resin (Midwest Biotech) was placed in areaction vessel and the following sequence was assembled on a CSBio336synthesizer using DEPBT/DIEA activated single couplings of Boc aminoacids.

Boc-HaibQGTFTSDYSKYLDERAAQDFVQWLLDGGPSSGAPPPSK(Fmoc)G-mbha resin

The Boc protected peptide resin was transferred to a manual reactionvessel and was treated with 20% piperidine/DMF at room temp for 10 min.The resin was filtered, washed with DMF 3 times, and an activatedsolution of Fmoc Lys(ivDde)-OH (EMD-Novabiochem) was added (previouslyprepared by dissolving 2 mmole Fmoc Lys(ivDde) in 4.0 ml 0.5M DEPBT/DMFand 0.35 ml (2 mmole) DIEA wad added.

The peptide resin was mixed at room temp for 16 hrs, then filtered,washed with DMF and was treated with 20% piperidine/DMF for 10 min. Theresin was filtered, washed with DMF several times, and an activatedsolution of Fmoc Glu-OBzl was added (previously prepared by dissolving 2mmole Fmoc Glu-OBzl in 4.0 ml 0.5M DEPBT/DMF and 0.35 ml DIEA wasadded). The reaction was mixed 1 hr at room temp then filtered and theresin washed with DMF. After another treatment with 20% pip/DMF, theresin was washed with DMF several times, and an activated solution ofpalmitic acid was added to complete the acylation of the α-amine of theside chain Lys at position 40 (previously prepared by dissolving 2 mmolepalmitic acid in 4.0 ml 0.5M DEPBT/DMF and adding 0.35 ml DIEA). Thereaction was mixed for 1 hr.

The peptide resin was filtered, washed with DMF and treated with 2%hydrazine/DMF at room temp for 15 min, the filtered and was washed withDMF several times. An activated solution of Fmoc Glu-OBzl was added(same as above) and the reaction mixed for 1 hr at rt. The resin wasdivided into three portions for final acylation at the ε-lysine amine.In this case, to one portion an activated solution of docecanoic acidwas added. Previously prepared by dissolving 1 mmole dodecanoic acid(Aldrich) in 2.0 ml 0.5M DEPBT/DMF and 0.175 ml DIEA was added. Thereaction was mixed 1 hr, the filtered, washed with DMF, then withdichloromethane. The N-terminal Boc group was removed on treatment with50% TFA/DCM, and after neutralization with 5% DIEA/DCM, an HF cleavagewas run in an ice bath for 1 hrs using p-cresol as a scavenger. Afterevaporation of the HF, the residue was suspended in ethyl ether and thepeptide/resin mixture was filtered and washed with ether. The peptidewas extracted into aqueous acetic acid and analyzed on HPLC. (4.6×50 mmZorbax SB-C8, 1 ml/min, 45° C., 214 nm (0.5 A) A=0.1% TFA, B=0.1%TFA/90% ACN. The remaining cleavage extract was loaded onto a 21.2×250mm Amberchrom XT20 column and a 0.1% TFA/acetonitrile gradient was runon a Pharmacia FPLC for purification.

Fractions 83-86 were combined, frozen and lyophilized to give 22.3 mg ofthe peptide of SEQ ID NO: 211 with a purity of 90%+. Theoreticalmol.wt.=5202.9, ESI observed mass=5202.0

Peptide Acylation Via Succinoylation

Succinoylated peptides are prepared as follows. Peptides are synthesizedon a solid support resin using either a CS Bio 4886 Peptide Synthesizeror Applied Biosystems 430A Peptide Synthesizer. In situ neutralizationchemistry is used as described by Schnolzer et al., Int. J. PeptideProtein Res. 40: 180-193 (1992). For succincoylated peptides, the targetamino acid residue to be acylated (e.g., position ten, relative to theamino acid position numbering of SEQ ID NO: 3) is substituted with an Nε-FMOC lysine residue. Treatment of the completed N-terminally BOCprotected peptide with 20% piperidine in DMF for 10 minutes removedFMOC/formyl groups. The resin is filtered, washed with DMF/DCM andre-suspended in DCM. Coupling to the free ε-amino Lys residue isachieved by coupling a ten-fold molar excess of n-hexadecylsuccinicanhydride (TCl) along with 4-dimethylaminopyridine. The resin is mixedovernight, filtered, washed with DCM and treated with 50% TFA/DCM toremove any side chain protecting groups and the N-terminal BOC. Peptideresins are neutralized with 5% DIEA/DMF, dried, and then are cleavedfrom the support using HF/p-cresol, 95:5, at 0° C. for one hour.Following ether extraction, a 5% HOAc solution is used to solvate thecrude peptide. A sample of the solution is then verified by HPLCanalysis. The remaining acetic acid solution is loaded onto a 10×250 mmAmberchrom XT20 column for purification. An aqueous TFA/ACN gradient isrun while collecting fraction and monitoring the UV at 214 nm. Purefractions are lyophilized, yielding solid peptides.

An example synthesis of a succinoylated peptide (Glucagon Aib2 E16 A18L27 D28 Cex K40(C16 succinoyl)G41-amide having the amino acid sequenceof SEQ ID NO: 156) is shown in Figure FIG. 3C and is further describedbelow:

0.28 gm (0.2 mmole) mbha-resin (Midwest Biotech) was placed in areaction vessel and the following sequence was assembled on a CSBio336synthesizer using DEPBT/DIEA activated single couplings.

Boc-HaibQGTFTSDYSKYLDERAAQDFVQWLLDGGPSSGAPPPSK(Fmoc)G-mbha resin

Approximately, one third of the Boc protected peptide resin wastransferred to a manual reaction vessel and was treated with 20%piperidine/DMF at room temp for 10 min. The resin was filtered, washedwith DMF 3 times, with dichloromethane twice and re-suspended in 10 mlDCM. 324 mg (1 mmole) n-hexadecylsuccinic anhydride (TCI) was addedalong with 2-3 mg of 4-dimethylaminopyridine (Aldrich).

The resin was mixed overnight at room temp before filtering, washingwith DCM twice, and treating with 50% TFA/DCM for 1-2 min. The resin wasfiltered, washed several times with DCM, neutralized by washing with 5%DIEA/DCM and transferred to a HF reaction vessel. An HF cleavage was runusing 5 ml liquid hydrogen fluoride and 0.5 ml p-cresol scavenger. Afterstirring 1 hr in an ice bath, the HF was removed in vacuo and theresidue suspended in ethyl ether. The suspension was filtered using asintered glass funnel, the solids washed with ether, and the peptideextracted into 15 ml 50% aqueous acetic acid. After analysis on HPLC(4.6×50 mm Zorbax SB-C8, 1 ml/min, 45° C., 214 nm, A=0.1% TFA, B=0.1%TFA/90% ACN, gradient=30% B to 90% B over 10 min), the cleavage extractwas loaded onto a 21.2×250 mm Amberchrom XT20 column for purification.An aq TFA/acvetonitrile gradient was run while collecting fractions andmonitoring the UV absorbance. Fractions 55-56 were identified as beingsingle component and were frozen and lyophilized. 32.5 mg was recoveredwith a purity of 90%+ (DLS-027-68B). Theoretical mol. wt.=4720.3, ESIobserved mass=4717.0

Peptide Alkylation (e.g., S-Alkylation)

An example synthesis of an alkylated (e.g., S-alkylated) peptide(Glucagon Aib2 E16 A18 L27 D29 Cex Cys40 (S-2 palmityl)amide having anamino acid sequence of SEQ ID NO: 164) is shown in FIG. 6 and is furtherdescribed below:

0.28 gm 0.2 mmole mbha-resin (Midwest Biotech) was placed in a CSBioreaction vessel and the following sequence was assembled on a CSBio336synthesizer using Boc amino acids and DEPBT/DIEA activated singlecouplings.

HaibQGTFTSDYSKYLDERAAQDFVQWLLDGGPSSGAPPPSC-amide

The peptide resin was transferred to an HF reaction vessel and an HFcleavage was run using 10 ml liquid hydrogen fluoride and 1 ml p-cresolscavenger in an ice bath for 1 hr. After evaporating the HF, the residuewas suspended in ethyl ether and the peptide and resin were filteredinto a sintered glass funnel. After washing with ethyl ether and quicklyair drying, the peptide was extracted into 50% aqueous acetic acid.After analysis via HPLC (4.6×50 mm Zorbax SB-C8, 1 ml/min, 45° C., 214nm, A=0.1% TFA, B=0.1% TFA/90% ACN, 30% to 90% B over 10 min), thecleavage extract was diluted 3-4 with water and was loaded onto a22.2×250 mm Amberchrom XT20 column for purification using aq TFA/CANgradient. An initial pool was re-purified over the same column to give79 mg of 95% purity. Theoretical mol.wt.=4313.7, ESI observedmass=4312.0

16 mg (3.7 μmole) of the above free thiol peptide was suspended in 1 mlmethanol and with stirring under a stream of nitrogen, 1.5 ul oftetramethylguandine in 1 ml methanol (MeOH) was added followed by 3 mg(7.8 μmole) 2-iodohexadecanoic acid (2-iodopalmitic acid) in 0.5 mgtetrahydrofuran (THF). 2-iodohexadecanoic acid was previously preparedby treating 2-bromohexadecanoic acid with potassium iodide in acetone(DLS-027-52). The alkylation reaction was warmed in a bath for 10 minduring which most of the solvent evaporated. The residue was dissolvedin aqueous acetic acid, analyzed by HPLC and was found to be morehydrophobic than the starting peptide. The remaining acetic acidsolution was loaded onto a 10×250 mm Amberchrom XT20 column forpurification. An aq.TFA/ACN gradient was run while collecting fractionsand monitoring the UV at 214 nm. Fractions 64-68 were combined, frozenand lyophilized to give 6.1 mg of material (the peptide of SEQ ID NO:164) with a purity of 90%+. Theoretical mol.wt=4568.1, MALDI observedmass=4568.79

When the peptide comprises a Lys residue instead of the Cys residue atthe C-terminus, the peptide may be made with the backbone Lys first. Thetarget amino acid residue to be N-alkylated is substituted with an Nε-FMOC lysine residue. Treating of the completed N-terminally BOCprotected peptide with 20% piperidine in DMF for 30 minutes removesFMOC/formyl groups from the Lys residue. A Cys residue is coupled to thefree ε-amino Lys residue. The Cys residue is then alkylated by reactionwith 2-iodopalmitic acid as described above.

Peptide Acylation with “miniPEG” Spacers

An example synthesis of an acylated peptide via a mini-PEG spacer (thepeptide having an amino acid sequence of SEQ ID NO: 89) is describedbelow:

0.28 gm (0.2 mmole) 4-methylbenzhydrylamine (mbha) resin (MidwestBiotech, Inc., Fishers, Ind.) was placed in a reaction vessel and thefollowing sequence was assembled on a CSBio336 synthesizer using Bocamino acids and3-(diethoxyphosphoryloxy)-3H-benzo[d][1,2,3]triazine-4-one/N,N-Diisopropylethylamine(DEPBT/DIEA) activated single couplings.

Boc-HaibQGTFTSDYSKYLDERAAQDFVQWLLDGGPSSGAPPPSK(Fmoc)G-mbha

Approximately, one third of the Boc protected peptide resin wastransferred to a manual reaction vessel and was treated with 20%piperidine/dimethylformamide at room temperature for 10 min. The resinwas filtered, washed with dimethylformamide (DMF) several times and anactivated solution of Fmoc amidoPEG4 was added (previously prepared bydissolving 487.5 mg 1.0 mmole N-Fmoc Amido dPEG4 Acid (PeptidesInternational, Louisville, Ky.) in 2.0 ml 0.5M DEPBT/DMF and adding0.175 ml 1 mmole diisopropylethylamine). The reaction was mixed at roomtemp for aprox. 1 hr.

The peptide resin was filtered washed with DMF, and again treated with20% piperidine/DMF for 10 min. The resin was filtered, washed with DMFseveral times, and an activated solution of Fmoc Glu-OBzl was added(previously prepared by dissolving 470 mg 1 mmole of Fmoc Glu-γ-OBzl(Aapptec, Louisville, Ky.) in 2.0 ml 0.5M DEPBT/DMF and adding by 0.175ml 1 mmole DIEA). The reaction was mixed at room temp for 1 hr.

The peptide resin was again filtered, washed with DMF, and treated with20% piperidine/DMF at room temp for 10 min. The resin was filtered,washed with DMF several times, and an activated solution of palmiticacid was added (previously prepared by dissolving 256 mg 1 mmolepalmitic acid (Sigma-Aldrich, St. Louis, Mo.) in 2.0 ml 0.5M DEPBT/DMFand adding 0.175 ml DIEA. The reaction was mixed at room temp for 1 hr.

Finally, the peptide resin was filtered, washed with DMF followed bydichloromethane and was treated with 50% TFA/DCM to remove theN-terminal Boc group. After 1-2 min, the resin was filtered, washed withDCM several times followed by a solution of 5% DIEA/DCM. The air driedpeptide resin was transferred to an HF reaction vessel and an HFcleavage was conducted using 5 ml liquid hydrogen fluoride and 0.5 mlp-cresol scavenger. After mixing 1 hr in an ice bath, the HF was removedin vacuo and the residue was suspended in ethyl ether. The peptide/resinmixture was filtered in a sintered glass funnel, washed with ethylether, and quickly air dried. The peptide was extracted into 10 ml 50%aqueous acetic acid which was analyzed by HPLC (4.6×50 mm Zorbax SB-C8,1 ml/min, 214 nm, A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=30% B to 90%B over 10 min). The remaining crude extract was diluted 3× with waterand was loaded onto a 22.2×250 mm Amberchrom XT20 column forpurification using an aqueous TFA/ACN gradient. An initial purificationpool was re-purified over the same column to give 19 mg of product witha purity of 90%+. Theoretical mass=5010.6, ESI observed mass was 5008.0.

The same procedure was repeated using different N-Fmoc Amido dPEG Acids.In one instance, N-Fmoc Amido dPEG8 Acid (Peptides International,Louisville, Ky.) was used and in another instance, N-Fmoc Amido dPEG2Acid (Peptides International, Louisville, Ky.) was used. The structuresof the acylated peptides are shown in FIGS. 12A-12C.

Peptide Dimerization—Disulfide Dimerization

An example synthesis of a disulfide dimer peptide is described below:

0.28 gm (0.2 mmole) mbha-resin (Midwest Biotech) was placed in a CSBioreaction vessel and the following sequence was assembled on a CSBio336synthesizer using Boc amino acids and DEPBT/DIEA activated singlecouplings.

Boc-HaibQGTFTSDYSKYLDERAAQDFVQWLLDGGPSSGAPPPSK(Fmoc)G-amide

The Boc protected peptide resin was transferred to a manual reactionvessel and was treated with 20% piperidine/DMF at room temp for 10 min.The resin was filtered, thoroughly washed with DMF, then acylated withan activated solution of Fmoc Cys(Trt) (previously prepared bydissolving 1.17 gm 2 mmole Fmoc Cys(Trt)-OH (Aapptec) in 4.0 ml 0.5MDEPBT/DMF and adding 0.35 ml 2 mmole diisopropylethylamine). Thereaction was mixed at room temp for 2 hrs, then filtered washed with DMFand the same procedure was used to add Fmoc Glu-OBzl followed bypalmitic acid.

Finally, the resin was filtered, washed with DMF, with dichlorormethane,and was treated with 50% TFA/DCM for 1-2 min. After neutralization with5% DIEA/DCM, the peptide resin was transferred to and HF reaction vesseland an HF cleavage was run using 10 ml liquid hydrogen fluoride and 1 mlp-cresol scavenger in an ice bath for 1 hr. After evaporating the HF,the residue was suspended in ethyl ether and the peptide and resin werefiltered into a sintered glass funnel. After washing with ethyl etherand quickly air drying, the peptide was extracted into 50% aqueousacetic acid. After analysis via HPLC (4.6×50 mm Zorbax SB-C8, 1 ml/min,45° C., 214 nm, A=0.1% TFA, B=0.1% TFA/90% ACN, 30% to 90% B over 10min), the cleavage extract was diluted 3-4 with water and was loadedonto a 21.2×250 mm Amberchrom XT20 column for purification using aqTFA/acetonitrile gradient. Purification fractions 64-67 were combined,frozen, and lyophilized to give 58.7 mg of material with a HPLC purityof 90%+.

DLS-027-97A Theoretical mol.wt.=4866.48, ESI observed mass=4864.0

HaibQGTFTSDYSKYLDERAAQDFVQWLLDGGPSSGAPPPSK(Cys(SH) γE-C16)G-amide

18.6 mg (3.8 μmole) of the above peptide was dissolved in 2.0 ml 3Mguanidine/0.05M tris (pH8.5) and 1 ml dimethylsulfoxide was added. Thereaction was stirred at room temp exposed to the air. After 6 hrs, ananalytical HPLC compared to the starting peptide showed the presence ofa more hydrophobic peak.

The reaction mixture was diluted with 20 ml 0.1% TFA and was loaded ontoa 10×250 mm Amberchrom XT20 column. A purification was run using a 0.1%TFA/acetonitrile gradient while monitoring the UV absorbance at 220 nm.Fractions 68-72 were combined and lyophilized to give 5.5 mg of purifieddimer.

HPLC purity was 90%+. Theoretical mol. wt.=9743.99, MALDI observedmass=9745.1. DLS-027-98B. The structure of the resulting dimer is shownin FIG. 9A.

Peptide Dimerization—Thioether Dimerization

An example synthesis of a thioether dimer is described below:

0.28 gm (0.2 mmole) mbha-resin (Midwest Biotech) was placed in areaction vessel and the following sequence was assembled on a CSBio336synthesizer using DEPBT/DIEA activated single couplings.

Boc-HaibQGTFTSDYSKYLDERAAQDFVQWLLDGGPSSGAPPPSK(Fmoc)G-mbha

One third of the Boc protected peptide resin was transferred to a manualreaction vessel and was treated with 20% piperidine/DMF at room temp for10 min. After washing several time with DMF, an activated solution ofBoc Dap(Fmoc) was added (previously prepared by dissolving 426 mg 1mmole Boc Dap(Fmoc)-OH (Chem-Impex) in 2.0 ml 0.5M DEPBT/DMF and 0.175ml 1 mmole DIEA was added). The reaction was mixed at room temp for 2hrs, the filtered, washed with DMF and re-treated with 20%piperidine/DMF as above. After washing with DMF, the resin was acylatedwith an activated solution of bromoacetic acid (previously prepared bydissolving 139 mg 1 mmole bromoacetic acid (Aldrich) in 2.0 ml 0.5MDEPBT/DMF and adding 0.175 ml DIEA). The reaction was mixed at room tempfor 1-2 hrs, then filtered and the resin washed with DMF followed byDCM. The peptide resin was treated with 50% TFA/DCM at room temp for 2min, then filtered, washed with DCM and neutralized with 5% DIEA/DCM.The completed peptide resin was transferred to an HF reaction vessel andan HF cleavage was conducted using 5 ml liquid hydrogen fluoride/0.5 mlp-cresol. After stirring 1 hr in an ice bath, the HF was evaporated andthe residue suspended in ethyl ether. The resin/peptide mixture wasfiltered into a sintered glass funnel and washed with ether. The peptidewas extracted into 50% aqueous acetic acid and the crude product wasanalyzed via HPLC: 4.6×50 mm Zorbax SB-C8, 1 ml/min, 45° C., 214 nm,A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=30% B to 90% B over 10 min. Thecleavage extract was loaded onto a 21.2×250 mm Amberchrom XT20 columnand a purification was run using an aqueous TFA/acetonitrile gradientwhile monitoring the UV absorbance at 220 nm. Fractions 48-53 werecombine frozen, and lyophilized to give 18 mg of the peptide of SEQ IDNO: 89 with K40(Dap-BrAcetyl), purity=90%+. Theoretical mol. wt.=4602.8,ESI observed mass=4616.0

HaibQGTFTSDYSKYLDERAAQDFVQWLLDGGPSSGAPPPSK(Dap-BrAcetyl)G-amide

15 mg (3.2 μmole) of the above K40(Dap-BrAcetyl) peptide and 15 mg ofthe peptide of SEQ ID NO: 89 K40(Cys γE-C₁₆) were dissolved in 3.0 ml 7Murea/0.05M tris (pH8.6) and was mixed at room temp while monitoring theHPLC of the reaction progress. After 30 min, most of the startingmaterials were reduced in peak height while a new peak was the majorcomponent. The reaction mixture was diluted with 25 ml 0.1% TFA and wasloaded onto a 10×250 mm Amberchrom XT20 column for purification. Anaqueous TFA/acetonitrile gradient was run while monitoring the UVabsorbance at 220 nm. Fractions 57-61 were combined, frozen, andlyophilized to give 7.1 mg of thioether dimer. HPLC purity=90%+,Theoretical mol.wt.=9401.4, MALDI observed mass=9402.8. The structure ofthe dimer is shown in FIG. 9B.

Peptide PEGylation

For peptide PEGylation, 40 kDa methoxy poly(ethylene glycol)idoacetamide (NOF) was reacted with a molar equivalent of peptide in 7MUrea, 50 mM Tris-HCl buffer using the minimal amount of solvent neededto dissolve both peptide and PEG into a clear solution (generally lessthan 2 mL for a reaction using 2-3 mg peptide). Vigorous stirring atroom temperature commenced for 4-6 hours and the reaction analyzed byanalytical RP-HPLC. PEGylated products appeared distinctly from thestarting material with decreased retention times. Purification wasperformed on a Vydac C4 column with conditions similar to those used forthe initial peptide purification. Elution occurred around buffer ratiosof 50:50. Fractions of pure PEGylated peptide were found andlyophilized. Yields were above 50%, varying per reaction.

Analysis Using Mass Spectrometry

The mass spectra were obtained using a Sciex API-III electrosprayquadrapole mass spectrometer with a standard ESI ion source. Ionizationconditions that were used are as follows: ESI in the positive-ion mode;ion spray voltage, 3.9 kV; orifice potential, 60 V. The nebulizing andcurtain gas used was nitrogen flow rate of 0.9 L/min. Mass spectra wererecorded from 600-1800 Thompsons at 0.5 Th per step and 2 msec dwelltime. The sample (about 1 mg/mL) was dissolved in 50% aqueousacetonitrile with 1% acetic acid and introduced by an external syringepump at the rate of 5 μL/min.

When the peptides were analyzed in PBS solution by ESI MS, they werefirst desalted using a ZipTip solid phase extraction tip containing 0.6μL C4 resin, according to instructions provided by the manufacturer(Millipore Corporation, Billerica, Mass., see the Millipore website ofthe world wide web at millipore.com/catalogue.nsf/docs/C5737).

High Performance Liquid Chromatography (HPLC) Analysis:

Preliminary analyses were performed with these crude peptides to get anapproximation of their relative conversion rates in Phosphate BufferedSaline (PBS) buffer (pH, 7.2) using high performance liquidchromatography (HPLC) and MALDI analysis. The crude peptide samples weredissolved in the PBS buffer at a concentration of 1 mg/ml. 1 ml of theresulting solution was stored in a 1.5 ml HPLC vial which was thensealed and incubated at 37° C. Aliquots of 1001 were drawn out atvarious time intervals, cooled to room temperature and analyzed by HPLC.

The HPLC analyses were performed using a Beckman System GoldChromatography system using a UV detector at 214 nm. HPLC analyses wereperformed on a 150 mm×4.6 mm C18 Vydac column. The flow rate was 1ml/min. Solvent A contained 0.1% TFA in distilled water, and solvent Bcontained 0.1% TFA in 90% CH3CN. A linear gradient was employed (40% to70% B in 15 minutes). The data were collected and analyzed using PeakSimple Chromatography software.

The initial rates of hydrolysis were used to measure the rate constantfor the dissociation of the respective prodrugs. The concentrations ofthe prodrug and the drug were estimated from their peak areasrespectively. The first order dissociation rate constants of theprodrugs were determined by plotting the logarithm of the concentrationof the prodrug at various time intervals. The slope of this plot givesthe rate constant ‘k’. The half lives of the degradation of the variousprodrugs were then calculated by using the formula t1/2=0.693/k.

Example 2

This example describes an exemplary method of testing the biologicalactivity of the peptides of the present disclosures, which methodinvolves assaying cAMP synthesis.

The ability of glucagon analogs to induce cAMP was measured in a fireflyluciferase-based reporter assay. HEK293 cells co-transfected with areceptor (glucagon receptor, GLP-1 receptor or GIP receptor) andluciferase gene linked to cAMP responsive element were serum deprived byculturing 16 h in DMEM (Invitrogen, Carlsbad, Calif.) supplemented with0.25% Bovine Growth Serum (HyClone, Logan, Utah) and then incubated withserial dilutions of either glucagon, GLP-1, GIP or novel glucagonanalogs for 5 h at 37° C., 5% CO₂ in 96 well poly-D-Lysine-coated“Biocoat” plates (BD Biosciences, San Jose, Calif.). At the end of theincubation 100 microliters of LucLite luminescence substrate reagent(Perkin-Elmer, Wellesley, Mass.) were added to each well. The plate wasshaken briefly, incubated 10 min in the dark and light output wasmeasured on MicroBeta-1450 liquid scintillation counter (Perkin-Elmer,Wellesley, Mass.). Effective 50% concentrations were calculated by usingOrigin software (OriginLab, Northampton, Mass.

Example 3

This example describes an exemplary method of assaying the stability ofpeptides of the present disclosures.

Each glucagon analog is dissolved in water or PBS and an initial HPLCanalysis is conducted. After adjusting the pH (4, 5, 6, 7), the samplesare incubated over a specified time period at 37° C. and are re-analyzedby HPLC to determine the integrity of the peptide. The concentration ofthe specific peptide of interest is determined and the percent remainingintact is calculated relative to the initial analysis.

Example 4

This example describes an exemplary method of assaying solubility ofpeptides.

A solution (1 mg/ml or 3 mg/ml) of glucagon (or an analog) is preparedin 0.01N HCl. 100 ul of stock solution is diluted to 1 ml with 0.01N HCland the UV absorbance (276 nm) is determined. The pH of the remainingstock solution is adjusted to pH7 using 200-250 ul 0.1M Na₂HPO₄ (pH9.2).The solution is allowed to stand overnight at 4° C. then centrifuged.100 ul of supernatant is then diluted to 1 ml with 0.01N HCl, and the UVabsorbance is determined (in duplicate).

The initial absorbance reading is compensated for the increase in volumeand the following calculation is used to establish percent solubility:

${\frac{{Final}\mspace{14mu} {Absorbance}}{{Initial}\mspace{14mu} {Absorbance}} \times 100} = {{percent}\mspace{14mu} {soluble}}$

Example 5

This example describes an exemplary method of assaying peptides forbinding to a receptor.

The affinity of peptides to the glucagon receptor is measured in acompetition binding assay utilizing scintillation proximity assaytechnology. Serial 3-fold dilutions of the peptides made inscintillation proximity assay buffer (0.05 M Tris-HCl, pH 7.5, 0.15 MNaCl, 0.1% w/v bovine serum albumin) are mixed in 96 well white/clearbottom plate (Corning Inc., Acton, Mass.) with 0.05 nM(3-[¹²⁵I]-iodotyrosyl) Tyr10 glucagon (Amersham Biosciences, Piscataway,N.J.), 1-6 micrograms per well, plasma membrane fragments prepared fromcells over-expressing human glucagon receptor, and 1 mg/wellpolyethyleneimine-treated wheat germ agglutinin type A scintillationproximity assay beads (Amersham Biosciences, Piscataway, N.J.). Upon 5min shaking at 800 rpm on a rotary shaker, the plate is incubated 12 hat room temperature and then is read on MicroBeta1450 liquidscintillation counter (Perkin-Elmer, Wellesley, Mass.). Non-specificallybound (NSB) radioactivity is measured in the wells with 4 times greaterconcentration of “cold” native ligand than the highest concentration intest samples and total bound radioactivity is detected in the wells withno competitor. Percent specific binding is calculated as following: %Specific Binding=((Bound-NSB)/(Total bound-NSB))×100. IC₅₀ values weredetermined by using Origin software (OriginLab, Northampton, Mass.).

Example 6

Each of the peptide of SEQ ID NO: 10 and the peptide of SEQ ID NO: 11was analyzed for points of chemical instability. Analogs of each peptidewere designed to enhance chemical stability, synthesized, and tested invitro for agonist activity at each of the GLP-1 receptor, glucagonreceptor, and GIP receptor as essentially described in Example 2.

The EC₅₀s at the GLP-1 receptor (GLP-1R), the glucagon receptor (GR),and the GIP receptor (GIPR) are provided in Table 1.

TABLE 1 EC50 at Glucagon EC50 at GLP- EC50 at GIP Receptor 1 ReceptorReceptor SEQ ID # (nM) (nM) (nM) 10 0.004 0.004 0.004 18 0.007 0.0080.007 43 0.006 0.004 0.005 44 1.470 0.007 0.027 11 0.004 0.001 0.024 150.005 0.003 0.008 16 0.028 0.002 0.054 17 0.019 0.008 0.020

Example 7

Analogs of SEQ ID NO: 18 were made as essentially described in Example 1and tested in vitro for agonist activity at each of the GLP-1 receptor,glucagon receptor, and GIP receptor as essentially described in Example2: The EC₅₀s at the GLP-1 receptor (GLP-1R), the glucagon receptor (GR),and the GIP receptor (GIPR) are provided in Table 2.

TABLE 2 Amino acids EC50 at at positions Glucagon EC50 at GLP- EC50 atGIP SEQ ID # 16-21 Receptor 1 Receptor Receptor 10 ERAAQD 0.006 0.0100.005 11 ERAAQD 0.011 0.013 0.019 18 ERAAQE 0.005 0.010 0.004 19 ERAAH0.012 0.018 0.015 20 ERAAKD 0.006 0.010 0.005 21 ERAAaibE 0.002 0.0040.001 22 ARAAaibE 0.005 0.011 0.004 23 ERAaibAE 0.261 0.007 0.013 24aibRAAaibE 0.002 0.010 0.005 25 HRAAaibE 0.005 0.004 0.003 26 EQAAaibE0.004 0.001 0.001 27 ERAaibKE 0.165 0.002 0.004 28 KHAAaibE 0.006 0.0020.001 aib = amino isobutyric acid

Example 8

Analogs of SEQ ID NO: 18 varying at position 3 were made as essentiallydescribed in Example 1 and tested in vitro for agonist activity at eachof the GLP-1 receptor, glucagon receptor, and GIP receptor asessentially described in Example 2: The EC₅₀s at the GLP-1 receptor(GLP-1R), the glucagon receptor (GR), and the GIP receptor (GIPR) areprovided in Table 3.

TABLE 3 EC50 at Position 3 Glucagon EC50 at GLP- EC50 at GIP amino acidSEQ ID # Receptor 1 Receptor Receptor Gln 10 0.006 0.010 0.005 Gln 180.005 0.010 0.004 Glu 29 1.501 0.008 0.017 Ile 30 Not active 0.250 0.423Met 31 0.030 0.002 0.005 Leu 32 Not active 0.388 0.730 Hse 33 0.0150.002 0.004 acetyl Orn 34 0.368 0.001 0.020 Ala 35 0.073 0.002 0.006Acetyl Dab 36 0.009 0.003 0.014 Val 37 0.029 0.002 0.002 NorVal 38 0.0550.002 0.004 acetyl Lys 39 0.543 0.010 0.336 Nle 40 0.068 0.002 0.004Met(O)₂ 41 0.592 0.002 0.067 Met(O) 42 0.042 0.001 0.006 Hse =homoserine; NorVal = norvaline; Met(O) = methionine sulfoxide; acetylOrn = N-acetyl ornithine, wherein the side chain nitrogen is acetylated;acetyl Dab = N-acetyl diaminobutanoic acid, wherein the side chainnitrogen is acetylated; acetyl Lys = N-acetyl lysine, wherein the sidechain nitrogen is acetylated; Met(O)₂ = methionine sulfone

Example 9

Analogs of SEQ ID NO: 43 varying at position 3 were made as essentiallydescribed in Example 1 and tested in vitro for agonist activity at eachof the GLP-1 receptor, glucagon receptor, and GIP receptor asessentially described in Example 2: The EC50s at the GLP-1 receptor(GLP-1R), the glucagon receptor (GR), and the GIP receptor (GIPR) areprovided in Table 4.

TABLE 4 EC50 at Position 3 Glucagon EC50 at GLP- EC50 at GIP amino acidSEQ ID # Receptor 1 Receptor Receptor Gln 10 0.006 0.010 0.005 Gln 430.005 0.010 0.004 Hse 48 0.016 0.003 0.004 NorVal 47 0.025 0.003 0.002Val 45 0.026 0.006 0.005 Met(O) 46 0.089 0.003 0.018 Hse = homoserine;NorVal = norvaline; Met(O) = methionine sulfoxide

Example 10

The in vivo effects of select peptides of Table 3 were evaluated bysubcutaneously administering 10 nmol/kg peptide or a vehicle control toDIO mice that either had or did not have the gene encoding the GLP-1receptor knocked out. The peptide was administered 3 times per week (onMondays, Wednesdays, and Fridays) for 2 weeks. The mice were given ahigh fat diet and each group of mice contained 8 animals per group. Theaverage body weight was −52 g and the mice were ˜8 months old males.

Body weight and food intake were measured on the days that peptide orvehicle control was administered to the mice. Fasting blood glucoselevels (6 hour fasting period) were measured once a week on days 0, 7,and 14. Some of the results are shown in FIG. 1.

As shown in FIG. 1, each of the tested peptides (peptide of SEQ ID NO:10, peptide of SEQ ID NO: 29, and peptide of SEQ ID NO: 18) caused atleast a 10% decrease in body weight in DIO mice that did not have thegene for the GLP-1 receptor knocked out by Day 10 and the weight losscontinued through Day 14.

Example 11

The in vivo effects of select peptides of Tables 3 and 4 were evaluatedby subcutaneously administering 10 nmol/kg peptide or a vehicle controlto DIO mice. The peptide was administered 3 times per week (on Mondays,Wednesdays, and Fridays) for 2 weeks. The mice were given a diabetogenicdiet and each group of mice contained 8 animals per group. The averagebody weight was ˜50 g and the mice were ˜6 months old males.

Body weight and food intake were measured on the days that peptide orvehicle control was administered to the mice. Body composition wasanalyzed on Days 0 and 14. Fasting blood glucose levels were measuredonce a week on days 0, 7, and 14. Data from this study are shown inFIGS. 2-5.

Example 12

Analogs of SEQ ID NO: 10 were made as essentially described in Example 1and tested in vitro for agonist activity at each of the GLP-1 receptor,glucagon receptor, and GIP receptor as essentially described in Example2: The EC50s at the GLP-1 receptor (GLP-1R), the glucagon receptor (GR),and the GIP receptor (GIPR) are provided in Table 2.

TABLE 2 Amino acid modification EC50 at (relative to Glucagon EC50 atGLP- EC50 at GIP SEQ ID # SEQ 10) Receptor 1 Receptor Receptor 10 None0.004 0.006 0.011 43 D28E 0.005 0.007 0.018 63 G29V 0.004 0.003 0.055 64G29A 0.002 0.003 0.019 65 G29aib 0.002 0.003 0.005 aib, amino isobutyricacid

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range and each endpoint, unless otherwise indicatedherein, and each separate value and endpoint is incorporated into thespecification as if it were individually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. An analog of glucagon (SEQ ID NO: 1), comprising: a. at position 1 anamino acid comprising an imidazole side chain, optionally, His; b. atposition 2 an DPP-IV protective amino acid, optionally, AIB; c. at anyof positions 9, 10, 12, 20, or 37-43 an acylated amino acid or alkylatedamino acid, optionally wherein the acyl or alkyl group is linked to theamino acid via a spacer; d. at position 20 an amino acid selected fromthe group consisting of: (i) an α,α-disubstituted amino acid; and (ii)an amino acid comprising an imidazole side chain; and e. up to tenadditional amino acid modifications relative to SEQ ID NO: 1; wherein,when the glucagon analog lacks a hydrophilic moiety, the glucagon analogexhibits a GIP percentage potency of at least 0.1%, wherein the glucagonanalog has less than 100-fold selectivity for the human GLP-1 receptorversus the GIP receptor. 2-11. (canceled)
 12. An analog of glucagon (SEQID NO: 1), comprising: a. an amino acid selected from the groupconsisting of: Ala, Leu, Ile, Val, norvaline, homoserine, Met, andmethionine sulfone, at position 3; b. an acylated amino acid oralkylated amino acid at any of positions 9, 10, 12, 16, 20, or 37-43,optionally wherein the acyl or alkyl group is linked to the amino acidvia a spacer; c. an alpha helix stabilizing amino acid at one or more ofpositions 16-21, and d. up to ten additional amino acid modificationsrelative to SEQ ID NO: 1; wherein, when the glucagon analog lacks ahydrophilic moiety, the glucagon analog exhibits a GIP percentagepotency of at least 0.1%, wherein the glucagon analog has less than100-fold selectivity for the human GLP-1 receptor versus the GIPreceptor. 13-41. (canceled)
 42. A peptide comprising (a) the sequence ofHX₂X₃GTFTSDX₁₀SKYLX₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁FVQWLX₂₇X₂₈X₂₉X₃₀PSS GX₃₅PPPS(SEQ ID NO: 66) wherein: X₂ is AIB; X₃ is Gln or an amino acid ofStructure I, II, or III; X₁₀ is Tyr or an amino acid covalently attachedto a C12 to C18 acyl or alkyl group; X₁₅ is an acidic amino acid,optionally, Glu or Asp; X₁₆ is any amino acid, optionally, any aminoacid other than Gly, Pro, and Ser; e.g., Glu, Ala, alpha,alpha-disubstituted amino acid (e.g., AIB), His, Lys, Glu, X₁₇ is Arg,His, Gln; X₁₈ is Arg or Ala; X₁₉ is Ala or alpha, alpha disubstitutedamino acid; X₂₀ is a selected from group consisting of: alpha,alpha-disubstituted amino acid (e.g., AIB) or Gln or His, Lys, or Ala;X₂₁ is an acidic amino acid, optionally, Asp or Glu; X₂₇ is Leu, Ala,Nle, or Met; X₂₈ is Ala or an acidic amino acid (optionally, Asp orGlu); X₂₉ is aliphatic, e.g., Ala or Gly or AIB or Val; X₃₀ is smallaliphatic amino acid, e.g., Ala or Gly X₃₅ is Ala or a basic amino acid(optionally, Arg or Lys); wherein, when X₂₈ is an acidic amino acid, X₃₅is a basic amino acid; optionally, wherein, when X₂₈ is Asp, then X₂₉ isnot Gly; wherein, when X₁₀ is Tyr, the peptide comprises at position 40an amino acid covalently attached to a C12 to C18 acyl or alkyl group,and, wherein, optionally, the peptide comprises Gly at position 41,wherein the C-terminal amino acid of the peptide is amidated; (b) thesequence SEQ ID NO: 66 with up to 3 amino acid modifications relative toSEQ ID NO: 66, wherein the peptide exhibits agonist activity at thehuman GIP receptor and, the human GLP-1 receptor and/or the humanglucagon receptor; (c) the sequence ofX₁X₂X₃GTFTSDX₁₀SKYLX₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁FVQWLX₂₇X₂₈X₂₉X₃₀PSS GX₃₅PPPS(SEQ ID NO: 67) wherein: X₁ is an amino acid comprising a side chainimidazole, optionally, a His or His derivative; X₂ is a DPP-IV protetiveamino acid, optionally, an alpha, alpha-disubstituted amino acid (e.g.,AIB); X₃ is selected from the group consisting of Glu, Ala, Leu, Ile,Nle, Val, NorVal, homoserine, Met, methionine sulfoxide, methioninesulfone, acetyl-Orn, acetyl-diaminobutanoic acid, acetyl-Lys; X₁₀ is anamino acid covalently attached to a C12 to C18 acyl or alkyl group: X₁₅is an acidic amino acid, optionally, Glu or Asp; X₁₆ is any amino acid,optionally, any amino acid other than Gly, Pro, and Ser; (e.g., Glu,Ala, alpha, alpha-disubstituted amino acid (e.g., AIB), His, Lys) X₁₇ isArg, His, Gln; X₁₈ is Arg or Ala; X₁₉ is Ala or alpha, alphadisubstituted amino acid; X₂₀ is a selected from group consisting of:alpha, alpha-disubstituted amino acid (e.g., AIB) or Gln or His, Lys, orAla; X₂₁ is an acidic amino acid, optionally, Asp or Glu; X₂₇ is Leu,Ala, Nle, or Met; X₂₈ is Ala or an acidic amino acid (optionally, Asp orGlu); X₂₉ is aliphatic, e.g., Ala or Gly or AIB or Val; X₃₀ is smallaliphatic amino acid, e.g., Ala or Gly X₃₅ is Ala or a basic amino acid(optionally, Arg or Lys); wherein, when X₂₈ is an acidic amino acid, X₃₅is a basic amino acid; optionally, wherein, when X₂₈ is Asp, then X₂₉ isnot Gly wherein, when X₁₀ is Tyr, the peptide comprises at position 40an amino acid covalently attached to a C12 to C18 acyl or alkyl group,and, wherein, optionally, the peptide comprises Gly at position 41,wherein the C-terminal amino acid of the peptide is amidated: (d) thesequence SEQ ID NO: 67 with up to 3 amino acid modifications relative toSEQ ID NO: 67, wherein the peptide exhibits agonist activity at thehuman GIP receptor and, the human GLP-1 receptor and/or the humanglucagon receptor; (e) the sequence of SEQ ID NO: 18; (f) a sequence ofSEQ ID NO: 18 with up to 3 amino acid modifications relative to SEQ IDNO: 18, wherein the peptide exhibits agonist activity at the human GIPreceptor and the human GLP-1 receptor; (g) the sequence of SEQ ID NO:43; (h) a sequence of SEQ ID NO: 43 with up to 3 amino acidmodifications relative to SEQ ID NO: 43, wherein the peptide exhibitsagonist activity at the human GIP receptor and the human GLP-1 receptor;or (i) a sequence of any one of SEQ ID NOs: 100-282 and 333-515. 43-99.(canceled)
 100. A dimer or multimer comprising two or more peptides ofclaim
 42. 101. A conjugate comprising the peptide of claim 42 and aconjugate moiety.
 102. (canceled)
 103. A pharmaceutical compositioncomprising a peptide of claim 42 and a pharmaceutically acceptablecarrier, diluent, or excipient.
 104. A method of reducing weight gain orinducing weight loss in a subject in need thereof, comprisingadministering to a patient in need thereof a pharmaceutical compositionof claim 103 in an amount effective to reduce weight gain or induceweight loss.
 105. (canceled)
 106. A method of treating diabetes,comprising administering to a patient in need thereof a pharmaceuticalcomposition of claim 103 in an amount effective to lower blood glucoselevels.
 107. (canceled)